U.S. patent application number 14/679723 was filed with the patent office on 2015-10-08 for controlled release of growth factors and signaling molecules for promoting angiogenesis.
The applicant listed for this patent is PRESIDENT AND FELLOWS OF HARVARD COLLEGE. Invention is credited to Lan Cao, David J. Mooney.
Application Number | 20150283210 14/679723 |
Document ID | / |
Family ID | 41377626 |
Filed Date | 2015-10-08 |
United States Patent
Application |
20150283210 |
Kind Code |
A1 |
Cao; Lan ; et al. |
October 8, 2015 |
Controlled Release Of Growth Factors And Signaling Molecules For
Promoting Angiogenesis
Abstract
The present invention comprises compositions, methods, and
devices for delivering angiogenic factors and signaling molecules
to a target tissue, and controlling the release of these factors
and signaling molecules to spatially and temporally restrict their
release and dissemination, for the purpose of promoting
angiogenesis in target tissues wherein increased blood supply is
needed.
Inventors: |
Cao; Lan; (Stoughton,
MA) ; Mooney; David J.; (Sudbury, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
PRESIDENT AND FELLOWS OF HARVARD COLLEGE |
Cambridge |
MA |
US |
|
|
Family ID: |
41377626 |
Appl. No.: |
14/679723 |
Filed: |
April 6, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12992617 |
Jan 6, 2011 |
9012399 |
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PCT/US2009/045856 |
Jun 1, 2009 |
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14679723 |
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61130486 |
May 30, 2008 |
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Current U.S.
Class: |
424/400 ;
514/8.1; 514/8.2 |
Current CPC
Class: |
A61L 2300/432 20130101;
A61L 2300/426 20130101; A61K 38/1858 20130101; A61K 38/1866
20130101; A61L 27/54 20130101; A61L 2300/412 20130101; A61P 9/00
20180101; A61L 2300/602 20130101; A61K 47/6903 20170801; A61L
2300/45 20130101; A61K 9/06 20130101; A61K 45/06 20130101; A61K
38/05 20130101; A61K 9/0019 20130101; A61L 2300/414 20130101 |
International
Class: |
A61K 38/18 20060101
A61K038/18; A61K 9/06 20060101 A61K009/06; A61K 38/05 20060101
A61K038/05; A61K 47/48 20060101 A61K047/48; A61K 9/00 20060101
A61K009/00 |
Goverment Interests
GOVERNMENT SUPPORT
[0002] This invention was made with Government support under RO1
HL069957 awarded by the National Institutes of Health. The
Government has certain rights in the invention.
Claims
1. A device comprising a scaffold composition, a bioactive
composition, and a regulatory agent, said bioactive composition and
said regulatory agent being incorporated into or coated onto said
scaffold composition, wherein said regulatory agent controls the
activity of said bioactive agent.
2. The device of claim 1, wherein said bioactive composition is
released from said scaffold composition at a first rate and said
regulatory agent is released from said scaffold composition at a
second rate.
3. The device of claim 1, wherein said bioactive composition is a
pro-angiogenic factor and said regulatory agent is an inhibitor or
enhancer of angiogenesis.
4. The device of claim 1, wherein said bioactive composition is a
vascular endothelial growth factor (VEGF) or a platelet derived
growth factor (PDGF).
5. The device of claim 1, wherein said regulatory agent is a Notch
inhibitor.
6. The device of claim 1, wherein said Notch inhibitor is
N-[N-(3,5-Difluorophenacetyl-L-alanyl)]-S-phenylglycine t-butyl
ester (DAPT).
7. The device of claim 1, wherein said device comprises a molar
ratio of 1:1 to 1:200 for VEGF.sub.165 to DAPT, 1:1 to 1:200 for
PDGF-BB to DAPT, and 1:0.1:1 to 1:10:200 for VEGF.sub.165, PDGF-BB
and DAPT, in an alginate polymer scaffold.
8. The device of claim 1, wherein said device comprises a molar
ratio of 1:31 for VEGF.sub.165 to DAPT, 1.8:31 for PDGF to DAPT,
and 1:1.8:31 for VEGF.sub.165:PDGF-BB:DAPT.
9. The device of claim 1, wherein said bioactive composition is
covalently linked to said scaffold composition.
10. The device of claim 1, wherein said bioactive composition is
non-covalently linked to said scaffold composition.
11. The device of claim 3, wherein said pro-angiogenic factor is
selected from the group consisting of ligands including endothelial
growth factor (VEGF (A-F)), fibroblast growth factors (acidic and
basic FGF 1-10), granulocyte-macrophage colony-stimulating factor
(GM-CSF), insulin, insulin growth factor or insulin-like growth
factor (IGF), insulin growth factor binding protein (IGFBP),
placenta growth factor (PIGF), angiopoietin (Ang1 and Ang2),
platelet-derived growth factor (PDGF), hepatocyte growth factor
(HGF), transforming growth factor (TGF-.alpha., TGF-.beta.,
isoforms 1-3), platelet-endothelial cell adhesion molecule-1
(PECAM-1), vascular endothelial cadherin (VE-cadherin), nitric
oxide (NO), chemokine (C--X--C motif) ligand 10 (CXCL10) or IP-10,
interleukin-8 (IL-8), hypoxia inducible factor (HIF), monocyte
chemotactic protein (MCP), vascular cell adhesion molecule (VCAM),
ephrin ligands (including Ephrin-B2 and -B4); transcription factors
including HIF-1.alpha., HIF-1.beta. and HIF-2.alpha., Ets-1, Hex,
Vezf1, Hox, GATA, LKLF, COUP-TFII, Hox, MEF2, Braf, Prx-1, Prx-2,
CRP2/SmLIM and GATA family members, basic helix-loop-helix factors
and their inhibitors of differentiation; and regulatory molecules
including enzymes (matrix metalloproteinase (MMP), tissue
plasminogen activator (PLAT or tPA), cyclooxygenase (COX),
angiogenin).
12. The device of claim 1, wherein said regulatory agent comprises
a signaling molecule.
13. The device of claim 12, wherein said signaling molecule is
selected from the group consisting of monoclonal antibodies to
Notch ligands and/or receptors, RNA interference of Notch ligands
or receptors, antisense Notch, receptor and mastermind-like 1
(MAML1) decoys, beta and gamma-secretase inhibitors (GSI), and a
molecule that activates or inhibits Notch signaling.
14. The device of claim 13, wherein said GSI is
N-[N-(3,5-Difluorophenacetyl-L-alanyl)]-S-phenylglycine t-butyl
ester (DAPT).
15. The device of claim 1, wherein the scaffold composition
degrades at a predetermined rate based on a physical parameter
selected from the group consisting of temperature, pH, hydration
status, and porosity.
16. The device of claim 1, wherein said scaffold composition is
enzymatically digested by a composition elicited by a contacting
cell, said release of said bioactive composition being dependent
upon the rate of enzymatic digestion.
17. A method of inducing growth of new blood vessels either from
existing blood vessels, or creation of de novo vessels, or a
combination thereof, in a target tissue of a mammal, comprising
contacting a mammalian cell with a device comprising a scaffold
composition with a bioactive composition and a regulatory agent
being incorporated therein or thereon and contacting a mammalian
tissue with said device, wherein said scaffold composition
temporally controls release of said bioactive composition and
wherein said bioactive composition induces angiogenesis,
arteriogenesis, or vasculogenesis.
18. The method of claim 17, wherein said bioactive composition
comprises VEGF or PDGF and wherein said regulatory agent comprises
DAPT.
19. The method of claim 17, wherein said bioactive composition is
released over a period of weeks and said regulatory agent is
released of a period of less than one week.
20. The method of claim 17, wherein a release rate of said
regulatory agent is 7-20 fold shorter than that of said bioactive
composition.
21. The method of claim 20, wherein said regulatory agent comprises
a Notch inhibitor and said bioactive composition comprises VEGF or
PDGF.
22. The method of claim 21, wherein said Notch inhibitor is
released within 1-3 days and said VEGF or PDGF is released within
7-60 days.
23. The method of claim 17, wherein said mammal is a human.
24. The method of claim 17, wherein said scaffold composition is
administered by injection, endoscopic delivery, minimally invasive
delivery, or surgical implantation.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of, and claims priority
under 35 U.S.C. .sctn.120 to, U.S. patent application Ser. No.
12/992,617 filed Jan. 6, 2011, which is a national stage
application, filed under 35 U.S.C. .sctn.371, of International
Application No. PCT/US2009/045856 filed Jun. 1, 2009, which claims
benefit under 35 U.S.C. .sctn.119 of U.S. Provisional Application
No. 61/130,486, filed May 30, 2008, the entire contents of each are
hereby incorporated herein by reference.
FIELD OF THE INVENTION
[0003] The present invention relates to the fields of cardiology,
tissue repair, and preventative medicine.
BACKGROUND OF THE INVENTION
[0004] Angiogenesis refers to a process of new blood vessel
formation. Subjects suffering from coronary arterial disease (CAD)
and peripheral arterial disease (PAD) can be treated by promoting
angiogenesis in the tissue lacking sufficient blood flow. However,
current methods of administering angiogenic drugs are sub-optimal
because they cannot control the presentation of multiple compounds
separately. Moreover, systemic administration of drugs at
concentrations that are therapeutically effective for the affected
area cause surrounding, healthy, tissues to be exposed
unnecessarily to pro-angiogenic growth factors and could lead to
undesirable side effects.
SUMMARY OF THE INVENTION
[0005] The present invention provides compositions and methods for
controlling the local presentations of pro-angiogenic growth
factors and signaling molecules that are together used to achieve
angiogenesis at the tissue or organ of interest. The compositions
and methods of the present invention allow the concerted and joint
presentations of delivered growth factors and signaling molecules
to be controlled separately by changing physical and/or chemical
properties of the polymer delivery material to achieve an
appropriate local concentration of the factor/molecules at the
target tissue site.
[0006] Subjects to be treated in the manner described herein have
been diagnosed as suffering from or at risk of developing an
ischemic condition. The methods are suitable to treatment of human
patients, as well as being suitable for veterinary use (e.g.,
treatment of companion animal such as dogs and cats. In a preferred
embodiment, the methods are used to develop treatments for chronic
ischemia in coronary and peripheral artery disease for diabetic
subjects. Alternatively, or in addition, the present invention is
used to improve wound healing in ulcers for diabetic subjects. The
impaired endothelial tissues of diabetic subjects often have a
reduced response to regular pro-angiogenic factors, making the
continuous activation of vascular growth induced by the sustained
and distinct presentation of growth factors and signaling molecules
provided by the present invention particularly valuable. This type
of continuous, sustained, and distinct presentation of
pro-angiogenic factors and signaling molecules is not currently
possible. Current methods that attempt to achieve similar
therapeutic outcomes also induce deleterious side-effects due to
substantial systemic dissemination of these factors throughout the
body.
[0007] A device, which overcomes the shortcomings of existing
approaches, comprises a scaffold composition, a bioactive
composition, and a regulatory agent. The bioactive composition and
the regulatory agent are incorporated into or coated onto the
scaffold, e.g., a polymeric gel, composition, and the regulatory
agent controls the activity of the bioactive agent. The bioactive
composition is released from the scaffold composition at a first
rate and the regulatory agent is released from said scaffold
composition at a second rate. For example, the bioactive
composition exits from the scaffold composition slower or faster
relative to the regulatory agent. For example, vascular endothelial
growth factor (VEGF) as a bioactive composition exits from the
scaffold composition for a time period of one or more, e.g., 4
weeks, while gamma secretase inhibitor as a regulatory agent exits
from the same scaffold composition for a time period of one or more
days, e.g., 3 days. The difference between the release rates of
multiple factors is important in determining the final angiogenesis
outcomes. For example, the desired time-frame for Notch inhibitor
delivery will be 7-20 fold (e.g., 8, 10, 15, or 18-fold) shorter
than that of VEGF or PDGF, i.e., Notch inhibitor is released within
1-3 days while VEGF or PDGF is released within 7-60 days (e.g., 7,
10, 15, 30, 45, or 60 days). An exemplary bioactive composition is
a pro-angiogenic factor such as VEGF and/or PDGF and an exemplary
regulatory agent is an inhibitor or enhancer of angiogenesis such
as DAPT.
[0008] One or more bioactive compositions are incorporated into or
coated onto the scaffold composition, and the scaffold composition
temporally controls release of the bioactive composition.
Alternatively, or in addition, the scaffold composition spatially
controls release of a bioactive composition. In another embodiment,
the bioactive composition incorporated into or coated onto the
scaffold composition temporally or spatially regulates release of a
second bioactive composition.
[0009] The present invention also comprises a method of inducing
blood vessel growth in a target tissue of a mammal, comprising
providing a device comprising a scaffold composition with a
bioactive composition being incorporated therein or thereon and
contacting a mammalian tissue with the device wherein said scaffold
composition temporally controls release of the bioactive
composition and wherein the bioactive composition induces
angiogenesis within the target tissue. For example, a polymeric gel
composition loaded with pro-angiogenic factors and a regulatory
factor is injected directly into the target site, or into a site
that is adjacent to or in close proximity to the target site in
which angiogenesis is desired. For example, the site of
administration is 10 mm, 25 mm, 50 mm, 1 cm, 5 cm, 10 cm, 50 cm
from the target tissue site where angiogenesis is to occur. In
addition, multiple simultaneous injections (in different spatial
locations, e.g., encircling or surrounding an affected anatomical
location/site), or repeating injections every 1-2 weeks, at the
same site or every few mm or cm apart in an ischemic region may be
desirable.
[0010] The present invention further comprises a method of
augmenting blood vessel growth, comprising providing a device
comprising a scaffold composition with a bioactive composition
being incorporated therein or thereon and contacting a mammalian
tissue with the device, wherein said scaffold composition
temporally controls release of the bioactive composition and
wherein the bioactive composition induces growth from existing
blood vessels.
[0011] The bioactive composition of the devices of the present
invention is non-covalently linked to said scaffold composition.
Alternatively, the bioactive composition is covalently linked to
said scaffold composition.
[0012] Bioactive compositions of the present invention consist of,
consist essentially of, or comprise one or more factors, which are
administered either by direct protein delivery or delivering gene
sequences to have cells locally make proteins. Exemplary bioactive
compositions can include receptor ligands, transcription factors,
and/or regulatory molecules.
[0013] Receptor ligands include, but are not limited to, vascular
endothelial growth factor (VEGF (A-F)), fibroblast growth factors
(acidic and basic FGF 1-10), granulocyte-macrophage
colony-stimulating factor (GM-CSF), insulin, insulin growth factor
or insulin-like growth factor (IGF), insulin growth factor binding
protein (IGFBP), placenta growth factor (PIGF), angiopoietin (Ang1
and Ang2), platelet-derived growth factor (PDGF), hepatocyte growth
factor (HGF), transforming growth factor (TGF-.alpha., TGF-.beta.,
isoforms 1-3), platelet-endothelial cell adhesion molecule-1
(PECAM-1), vascular endothelial cadherin (VE-cadherin), nitric
oxide (NO), chemokine (C--X--C motif) ligand 10 (CXCL10) or IP-10,
interleukin-8 (IL-8), hypoxia inducible factor (HIF), monocyte
chemotactic protein-1 (MCP-1), vascular cell adhesion molecule
(VCAM), ephrin ligands (including Ephrin-B2 and -B4). Transcription
factors include, but are not limited to, HIF-1.alpha., HIF-1.beta.
and HIF-2.alpha., Ets-1, Hex, Vezf1, Hox, GATA, LKLF, COUP-TFII,
Hox, MEF2, Braf, Prx-1, Prx-2, CRP2/SmLIM and GATA family members,
basic helix-loop-helix factors and their inhibitors of
differentiation.
[0014] Regulatory molecules include, but are not limited to,
enzymes (matrix metalloproteinase (MMP), tissue plasminogen
activator (PLAT or tPA), cyclooxygenase (COX), angiogenin),
molecules regulating Notch signaling which consists of, consists
essentially of, or comprises monoclonal antibodies to Notch ligands
and receptors, RNA interference, antisense Notch, receptor and
mastermind-like 1 (MAML1) decoys, beta and gamma-secretase
inhibitors (GSI), or any other molecules that can activate or
inhibit Notch signaling.
[0015] Devices of the present invention consist of, consist
essentially of, or comprise one or more bioactive compositions. A
second bioactive composition consists of, consists essentially of,
or comprises a signaling molecule selected from the group
consisting of monoclonal antibodies to Notch ligands and receptors,
RNA interference, antisense Notch, receptor and mastermind-like 1
(MAML1) decoys, beta and gamma-secretase inhibitors (GSI), or any
other molecules that can activate or inhibit Notch signaling.
[0016] Alternatively, or in addition, signaling molecules of the
second bioactive composition are selected from the group of any
other molecules that can inhibit or activate Notch signaling. In a
preferred embodiment of the present invention, the signaling
molecule is DAPT
(N-[N-(3,5-Difluorophenacetyl-L-alanyl)]-S-phenylglycine t-butyl
ester) (Sigma-Aldrich, St. Louis, Mo.). Signaling molecules of the
second bioactive composition are released from scaffolds and
devices simultaneously or sequentially with each other. Signaling
molecules of the second bioactive composition are released from
scaffolds and devices simultaneously or sequentially with bioactive
compositions comprising angiogenic factors. Preferably, the
regulatory molecule is a Notch inhibitor such as the gamma
secretase inhibitor DAPT.
[0017] Scaffold compositions of the present invention degrade at a
predetermined rate based on a physical parameter selected from the
group consisting of temperature, pH, hydration status, and
porosity. Alternatively, or in addition, scaffold compositions are
enzymatically digested by a composition elicited by a contacting
cell, said release of said bioactive composition being dependent
upon the rate of enzymatic digestion. A contacting cell is defined
as a cell belonging to a target cell wherein the scaffold
composition or device resides that physically contacts or adheres
to the scaffold composition or device.
[0018] Scaffold compositions of the present invention contain an
external surface. Alternatively, or in addition, scaffold
compositions contain an internal surface. External or internal
surfaces of the scaffold composition are solid or porous. Pore size
is less than about 10 nm, in the range of about 10 nm-20 .mu.m in
diameter, or greater than about 20 .mu.m. A scaffold composition
with multiple internal surfaces optionally comprises one or more
compartments.
[0019] Devices of the present invention are administered by
intramuscular injection. Alternatively, or in addition, scaffold
compositions and devices are administered by intraperitoneal
injection, or endoscopic delivery or other minimally invasive
delivery approach, or surgically implanted. Preferably, the loaded
gel composition is injected as a bolus using a standard syringe and
injection needle at or near the target angiogenesis site, or it can
be surgically implanted or delivered via a catheter.
[0020] The devices and methods of the invention provide a solution
to several problems associated with previous angiogenesis-inducing
protocols. The bioactive composition is incorporated into or coated
onto the scaffold composition. The scaffold composition and/or
bioactive composition temporally and spatially (directionally)
controls release of one or more additional bioactive
compositions.
[0021] This device includes a scaffold composition which
incorporates into or is coated with a bioactive composition; the
device releases one or more bioactive compositions comprised of
pro-angiogenic factors and signaling molecules to stimulate local
vascular growth. Release of the bioactive composition is regulated
spatially and temporally. Depending on the application for which
the device is designed, the device regulates release through the
physical or chemical characteristics of the scaffold itself. For
example, the scaffold composition is differentially permeable,
allowing release only in certain physical areas of the scaffold.
The permeability of the scaffold composition is regulated, for
example, by selecting or engineering a material for greater or
smaller pore size, density, polymer cross-linking, stiffness,
toughness, ductility, or viscoelasticity.
[0022] The scaffold composition contains physical channels or paths
through which bioactive compositions can move more easily towards a
targeted area of release of the device or of a compartment within
the device. The scaffold composition is optionally organized into
compartments or layers, each with a different permeability, so that
the time required for a bioactive composition to move through the
device is precisely and predictably controlled. Release is also
regulated by the degradation, de- or re-hydration, oxygenation,
chemical or pH alteration, or ongoing self-assembly of the scaffold
composition. These processes are driven by diffusion or catalyzed
by enzymes or other reactive chemicals.
[0023] Alternatively, or in addition, release is regulated by a
bioactive composition. By varying the concentration of
extracellular matrix components, adhesion molecules and other
bioactive compounds in different areas of the device, including
agents with means to create pores and enzymatically digest the
scaffold composition, the bioactive composition has means to
control the rate at which other elements of the same bioactive
composition or additional bioactive compositions escape, or are
released from the scaffold composition or device. The device
controls and directs the flow of bioactive compositions or elements
through its structure.
[0024] Chemical affinities are used to channel bioactive
compositions towards a specific area of release. By varying the
density and mixture of those bioactive substances, the device
controls the timing of the combination and release of elements of
bioactive compositions or multiple bioactive compositions. In one
embodiment, components of a bioactive composition or two
compositions are separated initially, but combined when allowed to
flow through channels in the scaffold composition towards an area
of release. The density and mixture of these bioactive substances
is controlled by initial doping levels or concentration gradient of
the substance, by embedding the bioactive substances in scaffold
material with a known leaching rate, by release as the scaffold
material degrades, by diffusion from an area of concentration, by
interaction of precursor chemicals diffusing into an area, or by
production/excretion of compositions by neighboring cells.
[0025] Cells in close physical proximity to the scaffold
compositions and devices of the present invention, or those in
physical contact with the scaffold composition and devices, secrete
enzymes that affect the one or more features of the scaffold
composition. Neighboring or juxtaposed cells residing within target
tissues increase or decrease the structural integrity of the
scaffold composition through directly (e.g., release of enzymes) or
indirectly (e.g. release of signals recruiting cells to the
scaffold compositions that affect structural integrity).
Neighboring or juxtaposed cells produce factors that increase or
decrease the rigidity, increase or decrease the porosity, increase
or decrease the potential for or rate of degradation, increase or
decrease the adhesion or mobility, or increase or decrease the
immunogenicity of the scaffold composition or device.
Alternatively, the scaffold composition is comprised of materials
that are unaffected by enzymatic activity and are unaffected by
cellular secretions from local tissues.
[0026] The physical or chemical structure of the scaffold also
regulates the diffusion of bioactive agents through the device. The
release profiles of multiple bioactive compositions are made
distinct from each other by adjusting the properties and
formulation of delivery vehicle. For example, the
small-molecule-weight signaling molecule is first incorporated into
microspheres followed by incorporation into alginate hydrogel, so
that the signaling molecules have a more delayed release compared
to the growth factors. The pore size, oxidization degree, molecular
weight distribution of the alginate gel are varied to control the
release rate of incorporated growth factors.
[0027] The bioactive composition includes one or more compounds
that regulate cell function and/or behavior. The bioactive
composition is covalently linked to the scaffold composition or
non-covalently associated with the scaffold. For example, the
bioactive composition is an extracellular matrix (ECM) component
that is chemically crosslinked to the scaffold composition.
Regardless of the tissue of origin, ECM components generally
include three general classes of macromolecules: collagens,
proteoglycans/glycosaminoglycans (PG/GAG), and glycoproteins, e.g.,
fibronectin (FN), laminin, and thrombospondin. ECM components
associate with molecules on the cell surface and mediate adhesion
and/or motility. Preferably, the ECM component associated with the
scaffold is a proteoglycan attachment peptide or cyclic peptide
containing the amino acid sequence arginine-glycine-aspartic acid
(RGD). Proteoglycan attachment peptides are selected from the group
consisting of G.sub.4RGDSP (SEQ ID NO: 1), XBBXBX (SEQ ID NO: 2),
PRRARV (SEQ ID NO: 3), YEKPGSPPREVVPRPRPGV (SEQ ID NO: 4),
RPSLAKKQRFRHRNRKGYRSQRGHSRGR (SEQ ID NO: 5), and RIQNLLKITNLRIKFVK
(SEQ ID NO: 6), and cell attachment peptides are selected from the
group consisting of RGD, RGDS, LDV, REDV, RGDV, LRGDN (SEQ ID NO:
7), IKVAV (SEQ ID NO: 8), YIGSR (SEQ ID NO: 9), PDSGR (SEQ ID NO:
10), RNIAEIIKDA (SEQ ID NO: 11), RGDT, DGEA, and VTXG.
[0028] Components of the ECM, e.g., FN, laminin, and collagen,
interact with the cell surface via the integrin family of
receptors, a group of divalent cation-dependent cell surface
glycoproteins that mediate cellular recognition and adhesion to
components of the ECM and to other cells. Ligands recognized by
integrins typically contain an RGD amino acid sequence that is
expressed in many ECM proteins. Exemplary molecules that mediate
cell adhesion and/or movement include FN, laminin, collagen,
thrombospondin 1, vitronectin, elastin, tenascin, aggrecan, agrin,
bone sialoprotein, cartilage matrix protein, fibrinogen, fibrin,
fibulin, mucins, entactin, osteopontin, plasminogen, restrictin,
serglycin, SPARC/osteonectin, versican, von Willebrand Factor,
polysaccharide heparin sulfate, cell adhesion molecules including
connexins, selectins include collagen, RGD (Arg-Gly-Asp) and YIGSR
(Tyr-Ile-Gly-Ser-Arg) peptides, glycosaminoglycans (GAGs),
hyaluronic acid (HA), integrins, selectins, cadherins and members
of the immunoglobulin superfamily. Carbohydrate ligands of the ECM
include the polysaccharides hyaluronic acid, and
chondroitin-6-sulfate.
[0029] The device optionally contains a second or third bioactive
composition(s), e.g., a growth factor, differentiation factor, or
signaling molecule. For example, the device includes vascular
endothelial growth factor (VEGF), hepatocyte growth factor (HGF),
or fibroblast growth factor 2 (FGF2) or a combination thereof.
Growth factors used to promote angiogenesis, wound healing, and
other aspects of tissue regeneration are listed herein and are used
alone or in combination to induce regeneration of bodily tissues by
bioactive compositions released from an implanted scaffold
device.
[0030] The bioactive composition(s) described above is
non-covalently linked to the scaffold composition. Alternatively,
or in addition, the bioactive composition is covalently associated
with the scaffold. Noncovalent bonds are generally one to three
orders of magnitude weaker than covalent bonds permitting diffusion
of the factor out of the scaffold and into surrounding tissues.
Noncovalent bonds include electrostatic, hydrogen, van der Waals,
.pi. aromatic, and hydrophobic. For example, a growth factor such
as VEGF is associated with the device by noncovalent bonds and
exits the device following administration of the device to a target
site to promote angiogenesis within the target bodily tissue.
[0031] The polymer scaffold composition into or onto which the
bioactive composition (growth factor, and/or regulatory molecule
are loaded is biocompatible. The composition is
bio-degradable/erodable or resistant to breakdown in the body.
Relatively permanent (degradation resistant) scaffold compositions
include metals and some polymers such as silk. Preferably, the
scaffold composition degrades at a predetermined rate based on a
physical parameter selected from the group consisting of
temperature, pH, hydration status, and porosity, the cross-link
density, type, and chemistry or the susceptibility of main chain
linkages to degradation or it degrades at a predetermined rate
based on a ratio of chemical polymers. For example, a high
molecular weight polymer comprised of solely lactide degrades over
a period of years, e.g., 1-2 years, while a low molecular weight
polymer comprised of a 50:50 mixture of lactide and glycolide
degrades in a matter of weeks, e.g., 1, 2, 3, 4, 6, 10 weeks. A
calcium cross-linked gels composed of high molecular weight, high
guluronic acid alginate degrade over several months (1, 2, 4, 6, 8,
10, 12 months) to years (1, 2, 5 years) in vivo, while a gel
comprised of low molecular weight alginate, and/or alginate that
has been partially oxidized, will degrade in a matter of weeks. A
typical volume of alginate gel is 1 .mu.L to 1 mL, with a
degradation time ranging from 1 day to 6 weeks.
[0032] In one example, cells mediate degradation of the scaffold
matrix, i.e., the scaffold composition is enzymatically digested by
a composition elicited by a neighboring cell, and the release of
the bioactive composition is dependent upon the rate of enzymatic
digestion of the scaffold. In this case, polymer main chains or
cross-links contain compositions, e.g., oligopeptides, which are
substrates for collagenase or plasmin, or other enzymes produced by
cells adjacent to the scaffold.
[0033] Exemplary scaffold compositions include polylactic acid,
polyglycolic acid, PLGA polymers, alginates and alginate
derivatives, gelatin, collagen, fibrin, hyaluronic acid, laminin
rich gels, agarose, natural and synthetic polysaccharides,
polyamino acids, polypeptides, polyesters, polyanhydrides,
polyphosphazines, poly(vinyl alcohols), poly(alkylene oxides),
poly(allylamines)(PAM), poly(acrylates), modified styrene polymers,
pluronic polyols, polyoxamers, poly(uronic acids),
poly(vinylpyrrolidone) and copolymers or graft copolymers of any of
the above. One preferred scaffold composition includes alginate
gels.
[0034] Porosity of the scaffold composition influences release of
one or more bioactive compositions from the device. Pores are
nanoporous, microporous, or macroporous. For example, the diameter
of nanopores are less than about 10 nm; micropore are in the range
of about 100 nm-20 .mu.m in diameter; and, macropores are greater
than about 20 .mu.m (preferably greater than about 100 .mu.m and
even more preferably greater than about 400 .mu.m). In one example,
the scaffold is macroporous with aligned pores of about 400-500
.mu.m in diameter.
[0035] In one preferred embodiment of the invention, one or more of
the bioactive compositions contains an element with means to
chemically induce pores in the scaffold composition that are
nanoporous, microporous, or macroporous in size. The abundance of
this element and the presence or absence of other elements that
augment or inhibit the activity of this pore-making element
determine pore size and density, and consequently, the rate at
which one or more bioactive compositions passively escape or are
actively released from the scaffold composition. Exemplary elements
with means to chemically induce pores in the scaffold composition
include, but are not limited to, potassium salts, calcium salts,
magnesium salts, amino acids, week acids, carbohydrates, potassium
bitartrate, creatine, aspargine, glutamine, aspartic acid, glutamic
acid, leucin, neroleucine, inosine, isoleucine, magnesium citrate,
magnesium phosphate, magnesium carbonate, magnesium hydroxide, and
magnesium oxide. Pore-forming elements are also enzymes
incorporated into or coated onto the scaffold composition. One or
more of these elements are initially contained within separate
compartments of the scaffold composition and later combined by
having one or more of these elements flow through common channels
or paths in the scaffold composition. These elements individually
have means to induce pore formation in the scaffold composition.
Alternatively, these elements are combined, and the resulting
combination has means to induce pore formation in the scaffold
composition.
[0036] The devices are manufactured in their entirety in the
absence of cells or can be assembled around or in contact with
cells (the material is gelled or assembled around cells in vitro or
in vivo in the presence of cells and tissues). In one embodiment of
the invention, the scaffold composition material with one or more
bioactive compositions incorporated, is injected into a target
tissue where local blood flow is restricted or would healing is
impaired.
[0037] The device is manufactured in one stage comprising one layer
or compartment. Alternatively, the device is manufactured in two or
more (3, 4, 5, 6, . . . 10 or more) stages in which one layer or
compartment is made and infused or coated with a bioactive
composition followed by the construction of a second, third, fourth
or more layers, which are in turn infused or coated with a
bioactive composition in sequence. Each layer or compartment is
identical to the others or distinguished from one another by the
elements comprising the bioactive composition incorporated into or
coated onto them as well as distinct chemical, physical and
biological properties.
[0038] A method of making a scaffold is carried out by providing a
scaffold composition and covalently linking or noncovalently
associating the scaffold composition with a first bioactive
composition. The scaffold composition is also contacted with a
second bioactive composition. The second bioactive composition is
non-covalently associated with the scaffold composition to yield a
doped (loaded) scaffold, i.e., a scaffold composition that includes
one or more bioactive substances. The contacting steps are
optionally repeated to yield a plurality of doped scaffolds, e.g.,
each of the contacting steps is characterized by a different amount
of the second bioactive composition to yield a gradient of the
second bioactive composition in the scaffold device. Rather than
altering the amount of composition, subsequent contacting steps
involve a different bioactive composition, i.e., a third, fourth,
fifth, sixth . . . , composition or mixture of compositions, that
is distinguished from the prior compositions or mixtures of prior
doping steps by the structure or chemical formula of the factor(s).
The method optionally involves adhering individual niches, layers,
or components to one another and/or insertion of semi-permeable,
permeable, or nonpermeable membranes within or at one or more
boundaries of the device to further control/regulate locomotion of
cells or bioactive compositions.
[0039] Therapeutic applications of the device include vascular
tissue generation, regeneration and repair. A mammalian tissue is
contacted with the device. The scaffold composition and/or the
bioactive composition spatially or directionally regulates release
of a bioactive composition with means to promote angiogenesis in
local tissues such as vascular, muscle, gastrointestinal, e.g.,
bowel, cardiac, brain, kidney, bone, nerve both central and
peripheral nervous system (CNS and PNS) or any tissue characterized
by a shortage of oxygen or nutrients or in which the vasculature is
damaged, absent, or functionally impaired.
[0040] A method of inducing local angiogenesis in a target tissue
is carried out by administering to a mammal a device containing a
scaffold composition and a bioactive composition incorporated
therein or thereon. The scaffold composition and/or bioactive
composition induces release of pro-angiogenic factors and signaling
molecules from the device into the local tissue environment. The
release of these angiogenic factors is controlled spatially and
temporally. Release can be sustained at a steady rate/dosage for a
desired period of time, e.g., minutes; 0.2. 0.5, 1, 2, 4, 6, 12, 24
hours; 2, 4, 6, days; weeks (1-4), months (2, 4, 6, 8, 10, 12) or
years, during which the cells are exposed to structural elements
and bioactive compositions that lead to improved vascular
growth.
[0041] Other features and advantages of the invention will be
apparent from the following description of the preferred
embodiments thereof, and from the claims. All references cited
herein are incorporated by reference.
BRIEF DESCRIPTION OF THE DRAWINGS
[0042] FIG. 1 is a bar graph showing the effect of VEGF on
sprouting ratio. An in vitro model was used to test the
significance of a controlled local concentration of VEGF.
[0043] FIG. 2 is a bar graph showing the effect of VEGF and GSI on
sprouting ratio. In vitro model was used to test the significance
of a combination of DAPT and VEGF.
[0044] FIG. 3 is a bar graph showing the effect of different
amounts and ratios of amounts of VEGF and GSI on sprouting ratio.
In vitro model establishing the significance of a distinct
presentation of VEGF and DAPT.
[0045] FIG. 4A is a line graph showing the in vitro release
profiles of VEGF and DAPT from injected alginate hydrogel
system.
[0046] FIG. 4B is a line graph showing the effect of VEGF and a GSI
(DAPT) on angiogenesis in an in vivo model testing the effect of
controlled presentation of VEGF and DAPT to blood flow recovery in
an ischemia situation. Blood flow recovery subject to hindlimb
ligation.
[0047] FIG. 4C is a line graph showing the effect of VEGF and a GSI
(DAPT) on angiogenesis in an in vivo model testing the effect of
controlled presentation of VEGF and DAPT to blood flow recovery in
an ischemia situation. Blood flow recovery subject to hindlimb
ligation. gel: represents the use of the alginate as the delivery
vehicle. im: intramuscular injection. ip: intraperitoneal
injection.
[0048] FIG. 5A is a bar graph showing the effect of VEGF and DAPT
on blood capillary density. An in vivo model was used to test the
effect of controlled presentation of VEGF and DAPT on newly formed
blood vessel density in an ischemia situation. +: use of the
substance. -: lack of the substance. gel: alginate gel.
[0049] FIG. 5B is a bar graph showing the effect of VEGF and DAPT
on blood capillary density. An in vivo model was used to test the
effect of controlled presentation of VEGF and DAPT on newly formed
blood vessel density in an ischemia situation. +: use of the
substance. -: lack of the substance. gel: alginate gel. im:
intramuscular injection. ip: intraperitoneal injection.
[0050] FIG. 6 is a series of photomicrographs showing the effect of
DAPT on gastrointestinal tissue. In vivo model was used to test the
effect of DAPT delivered from alginate gel system and from
intraperitoneal injection on the cells in small intestines. gel:
alginate gel. ip: intraperitoneal injection. H&E, alcian blue,
Ki67 and HES-1 are the four different staining methods to
characterize the crypt cells in small intestines. Ki-67 was used to
stain the proliferative cells. Loss of Notch signaling can alter
the proliferation rate of crypt cells. HES-1 staining was to
examine the expression of a known Notch target gene in crypts.
Alcian blue staining was to examine the deposition of
glycosaminoglycan molecules. Loss of Notch signaling can result in
more deposition of glycosaminoglycan molecules. Expression of HES-1
(hairy and enhancer of split 1), a member of basic helix-loop-helix
family of transcription factors and a known Notch target gene in
crypts was examined. Loss of Notch signaling can alter the
proliferation rate of crypt cells, as shown by Ki-67 staining the
proliferative cells. In addition, Notch inhibition has been
reported to alter the balance between proliferative crypt cells and
goblet cells, resulting in more deposition of glycosaminoglycan
molecules, as characterized by alcian blue staining. Notch
inhibition can also result in a significant alteration of the
morphology of the small intestine as compared to controls, as
demonstrated by hematoxylin and eosin (H&E) staining.
[0051] FIG. 7A is a bar graph showing the effect of VEGF, DAPT, and
the combination on blood capillary density.
[0052] FIG. 7B is a line graph showing the effect of VEGF, DAPT,
and the combination on blood flow.
[0053] FIG. 8A is a bar graph showing the effect of VEGF, PDGF, and
the combination on blood capillary density.
[0054] FIG. 8B is a line graph showing the effect of VEGF, PDGF,
and the combination on blood flow.
[0055] FIG. 9A is a bar graph showing the effect of VEGF, DAPT,
PDGF, and combinations thereof on blood capillary density.
[0056] FIG. 9B is a line graph showing the effect of VEGF, DAPT,
PDGF, and combinations thereof on blood flow.
[0057] FIG. 10A is series of photomicrographs showing the effect of
VEGF, DAPT, PDGF, and combinations thereof on maturation of newly
formed blood vessels in the local muscle tissues around the
ischemic site.
[0058] FIG. 10B is a bar graph showing the effect of VEGF, DAPT,
PDGF, and combinations thereof on maturation of newly formed blood
vessels as measured by density of smooth muscle actin (SMA)
positive vessels per unit area.
[0059] FIG. 10C is a bar graph showing the effect of VEGF, DAPT,
PDGF, and combinations thereof on maturation of newly formed blood
vessels as measured by the percentage of SMA positive vessels.
DETAILED DESCRIPTION OF THE INVENTION
[0060] The use of compounds that can modulate the signaling of
growth factors, e.g., gamma-secretase inhibitors (GSI), to promote
angiogenesis, has been proposed (US 2006/0264380 A1). However,
these previous studies did not indicate a need to provide the
compounds over particular time-frames, nor do they provide any
method to achieve a sustained presence of the compounds. Previous
studies mention the use of a combination of pro-angiogenic factors
together with GSI. Functionally, a bolus injection of multiple
pro-angiogenesis compounds simultaneously cannot control the
presentation of each individual compound separately, as they may
each need to have a distinct presentation. More importantly, single
or multiple injections that introduce drugs into system circulation
represent a sub-optimal method to reach the therapeutic level at
the specific tissue of interest, and can result in high
concentrations of drug accumulation at distant organs or tissues,
which may lead to various side effects.
[0061] The current invention provides a method to control the local
presentations of pro-angiogenic growth factors and signaling
molecules that are together used to achieve angiogenesis at the
tissue or organ of interest. The spatial and temporal presentation
of delivered growth factors and signaling molecules can be
controlled separately by fine-tuning the physical and chemical
properties of the polymer delivery material.
[0062] Specifically, this invention will be especially useful for
developing treatments for chronic ischemia in coronary or
peripheral artery disease for diabetics, or wound healing in
diabetes ulcers, as the impaired endothelium in these patients
normally have a reduced response to regular pro-angiogenesis
factors, thus a continuous activation by the sustained and distinct
presentation of growth factors and signaling molecules may be
critical. This is currently not achievable by other methods without
inducing possible side-effects.
[0063] Angiogenesis refers to a process of new blood vessel
formation. Patients suffering from coronary arterial disease (CAD)
and peripheral arterial disease (PAD) can be treated by promoting
angiogenesis in the tissue lacking sufficient blood blow. To
deliver compounds to induce angiogenesis is therefore a promising
therapeutic approach. Many growth factors, (e.g. vascular
endothelial growth factor (VEGF), play a vital role in inducing
angiogenesis by mediating the proliferation, migration and
differentiation of endothelial cells. Molecules that can modulate
the signaling pathways of growth factors, such as Notch inhibitors
(e.g., gamma secretase inhibitors (GSI)), can also be used to
augment the angiogenesis process. Therefore, delivering growth
factors together with molecules mediating signaling pathways may
have a beneficial effect.
[0064] Current delivery approaches mainly rely on injection of
factors/molecules alone, i.e., in the absence of a scaffold
composition. However, single injection is insufficient for
circumstances where growth factors and signaling molecules need to
be present over long time-frames, as they both have a short
half-life. In addition, bolus injection of multiple compounds
simultaneously cannot control the existence of each individual
compound separately, if they each need to have a distinct
presentation. More importantly, single or multiple injections that
introduce drugs into system circulation are sub-optimal to reach
the therapeutic level at the specific tissue of interest, and can
result in high concentrations at distant organs or tissues, which
may lead to various side effects.
[0065] The current invention utilizes an injectable biocompatible
polymer material system incorporating pro-angiogenic growth
factors, together with molecules that modulate the signaling
pathway, to achieve angiogenesis at the tissue or organ of
interest. The spatial and temporal presentation of delivered growth
factor and signaling molecules can be controlled separately by
fine-tuning the physical and chemical properties of the polymer
material.
Angiogenesis
[0066] Angiogenesis is a physiological process wherein new blood
vessels arise or extend from pre-existing vessels. The present
invention encompasses methods of promoting blood vessel growth from
existing vessels, as well as spontaneous blood vessel growth (also
referred to as vasculogenesis) and arteriogenesis (collateral
vessel formation). The term "angiogenesis" is meant to encompass
all three methods of blood vessel formation named supra.
[0067] Angiogenesis is a normal process in growth and development,
as well as in wound healing. Compositions and methods of the
present invention induce or augment endogenous mechanisms for
regulating angiogenesis and/or wound healing. Alternatively, or in
addition, compositions and methods of the present invention
introduce exogenous mechanisms for inducing or regulating
angiogenesis and/or wound healing that do not normally occur in a
target tissue. Furthermore, compositions and methods of the present
invention induce, regulate, augment, or replace mechanisms for
inducing or regulating angiogenesis and/or wound healing that are
insufficient, aberrant, or incomplete compared to the endogenous
mechanism due to genetic mutation, disease, infection, drug
treatment, medical condition, or tissue transplant procedure.
[0068] Angiogenesis is also a fundamental step in the transition of
neoplastic tumors from a dormant, or benign, state to a malignant,
cancerous, or metastatic, state. Compositions and methods of the
present invention disperse angiogenic factors and signaling
molecules within a local tissue environment and do not allow for
systemic administration or diffusion of bioactive compositions.
This ability of the devices of the present invention to contain
angiogenic factors to a confined target region demonstrates a
significant clinical advantage over previous methods of delivery
that are systemic in nature.
Controlled Release of Factors to Promote Angiogenesis
[0069] The release profiles of bioactive substances from scaffold
devices is controlled by both factor diffusion and polymer
degradation, the dose of the factor loaded in the system, and the
composition of the polymer. Similarly, the range of action (tissue
distribution) and duration of action, or spatiotemporal gradients
of the released factors are regulated by these variables. The
diffusion and degradation of the factors in the tissue of interest
is optionally regulated by chemically modifying the factors (e.g.,
PEGylating growth factors). In both cases, the time frame of
release determines the time over which effective angiogenesis by
the device is desired.
[0070] In the current system, the degradation rate of alginate, the
polymer carrier, is controlled by its composition (for example,
content of guluronic acid of alginate molecules, differential
molecular weight distribution of alginate), physical and chemical
treatment (e.g., irradiation or oxidization), and the degree of
crosslinking which is controlled by the choice and the amount of
the crosslinking agents (for e.g., ionic crosslinker or covalent
crosslinker). More specifically, increasing the content of
guluronic acid of alginate molecules will increase cross-linking
and slow degradation, decreasing molecular weight will speed up
degradation, irradiation will decrease molecular weight and thus
increase the degradation rate, oxidization will increase the
degradation rate, increasing the amount of crosslinker will slow
the degradation rate, and different crosslinker molecules may
result in differential crosslinking degree and affect the
degradation rate.
[0071] The doses of the factors loaded in the alginate carrier are
altered to achieve effective doses at a desired tissue site, e.g.,
from 1 to 10 microgram for VEGF.sub.165, 1 to 10 microgram for
PDGF-BB (dimeric glycoprotein composed of two B (-BB) chains), and
0.01 to 1 microgram for DAPT, for a mouse with an average weight of
10-50 g. An effective dosage amount or ratio of amounts is one that
induces and promotes angiogenesis at the target tissue or organ
site. The optimal dose is 3 to 10 microgram for VEGF.sub.165, 3 to
10 microgram for PDGF-BB, 0.05 to 1 microgram for DAPT in the
presence of VEGF at a dose of 1 to 10 microgram, and 0.05 to 1
microgram for DAPT in the presence of PDGF-BB at a dose of 1 to 10
microgram, for a mouse with an average weight of 10-50 g. The
amounts and ratio of amounts scale up proportionately for
humans.
[0072] The relative ratio of VEGF, PDGF and the Notch signaling
molecule (e.g., DAPT) is critical in determining the final outcomes
of angiogenesis. There exists an effective range and optimal value
of the relative ratio between the amount of VEGF and DAPT, the
amount of PDGF and DAPT, and the amount of VEGF, PDGF and DAPT. The
effective range of the relative ratio (by mole) is 1:1 to 1:200 for
VEGF.sub.165 to DAPT, 1:1 to 1:200 for PDGF-BB to DAPT, and 1:0.1:1
to 1:10:200 for VEGF.sub.165, PDGF-BB and DAPT, in the alginate
polymer system. The optimal relative ratio (by mole) is 1:31 for
VEGF.sub.165 to DAPT, 1.8:31 for PDGF to DAPT, and 1:1.8:31 for
VEGF.sub.165:PDGF-BB:DAPT. The optimal ratio may vary depending on
the specific delivery systems to be used, the species model (e.g.,
rat, rabbit, pig, dog, human and etc), and any change of the
composition and the release kinetics of each of these factors.
Arteriosclerosis
[0073] Arteriosclerosis, or arterial disease, is a general term
used to describe the thickening and hardening of the arteries. One
particular kind of arteriosclerosis that contributes to heart
disease is atherosclerosis. Atherosclerosis is a progressive
disease that is characterized by a buildup of plaque within the
arteries that may partially, or totally, block blood flow through
an artery. Plaque is formed from fatty substances, cholesterol,
cellular waste, calcium, and fibrin. Atherosclerosis generally
results in ischemia, or restriction of the blood supply, for the
tissues supplied by the blood carried in the blocked artery.
Oxygen Supply and Deprivation
[0074] Because oxygen is mainly bound to hemoglobin in red blood
cells, insufficient blood supply causes tissue to become hypoxic,
or, in more severe situations, when no oxygen is supplied, anoxic.
Oxygen deprivation can cause necrosis, or cell death. In very
aerobic tissues such as heart and brain, at body temperature,
necrosis due to ischemia becomes irreversible in 3-4 hours.
Complete oxygen deprivation to organs such as the heart and brain
for greater than 20 minutes causes irreversible damage.
[0075] Ischemia is a consequence of heart diseases, transient
ischemic attacks, cerebrovascular accidents, ruptured arteriovenous
malformations, and peripheral artery occlusive disease. The heart,
kidneys, and brain are the most sensitive organs to inadequate
blood supply. Stroke, aneurism, hemorrhage, and traumatic injury of
the brain commonly result in ischemic conditions. Ischemia in brain
tissue induces the ischemic cascade, in which proteolytic enzymes,
reactive oxygen species, and other harmful chemicals damage and may
ultimately kill brain tissue. Similarly, artery disease and
blockages can occlude blood flow to the heart inducing ischemia and
death of heart muscle tissue. A macroscopic region of necrotic
cells is called an infarction. Heart attacks lead to significant
cell death from prolonged oxygen deprivation, also referred to as
myocardial infarction.
[0076] Restoration of blood flow after a period of ischemia may
cause more damage than the ischemia. Reintroduction of oxygen
causes a greater production of damaging free radicals, resulting in
reperfusion injury and accelerated necrosis.
Coronary Arterial Disease
[0077] Patients diagnosed with Coronary Arterial Disease (CAD, also
called coronary heart disease, coronary artery disease, ischaemic
heart disease, and atherosclerotic heart disease) results from the
accumulation of atheromatous plaques within the walls of the
arteries that supply the myocardium (heart muscle) with oxygen and
nutrients. CAD encompasses a wide spectrum of patients with varying
disease severity and prognosis. Patients with mild CAD and the best
prognoses are asymptomatic. Mild CAD individuals have atheromatous
streaks within the walls of their coronary arteries that do not
obstruct blood flow and the lumen of their coronary artery is
normal in calibre (as assessed by coronary angiogram). As an
individual progresses along this spectrum toward more severe
phenotypes, the atheromatous streaks along the coronary walls
increase in thickness. Atheromatous plaques begin to form initially
and expand into the walls of the artery but, ultimately, expand
into the lumen of the vessel where they will begin to restrict
blood flow.
[0078] Once the plaques obstruct more than 70% of the diameter of
the vessel lumen, the individual develops symptoms of obstructive
coronary artery disease and is diagnosed with ischemic heart
disease. The first symptoms of ischemic heart disease are often
exertional angina (chest pain) or decreased exercise tolerance.
Angina that occurs regularly with activity, upon awakening, or at
other predictable times is termed stable angina. Angina that
changes in intensity, character or frequency is termed unstable.
Unstable angina may precede myocardial infarction.
[0079] The degree of severity of CAD can progress to near-complete
or complete blockage of the coronary artery. At this end of the
spectrum, most individuals experience one or more heart attacks
(myocardial infarctions) and all experience chronic ischemia. If
the blood flow to the heart tissue is restored to any degree,
ischemic tissue is capable of a partial or full recovery depending
upon the degree of blood flow restoration. Tissue that has suffered
from an infarction is dead, and the damage is irreversible.
Peripheral Arterial Disease
[0080] Peripheral Arterial Disease (PAD, also called peripheral
artery occlusive disease (PAOD), peripheral vascular disease, and
peripheral artery disease) is caused by the obstruction of large
peripheral arteries, which can result from atherosclerosis or
inflammatory processes and can lead to a narrowing of the artery
(stenosis) or obstruction of the artery by thrombus (obstruction by
blood clot) or embolism (obstruction by object carried in blood
stream from alternate location). PAD/PAOD results in ischemia that
is either acute (rapid onset, short duration) or chronic
(long-term). Exemplary symptoms of PAD/PAOD include, but are not
limited to, claudication (pain, weakness, or cramping in muscles
due to decreased blood flow); sores, wounds, or ulcers that heal
slowly or incompletely; change in color (blueness, paleness) or
cooling compared to other limbs; diminished hair or nail growth on
affected limbs compared to unaffected limbs.
[0081] PAD/PAOD occurrence is often associated with or caused by
smoking, diabetes mellitus, dyslipidemia (e.g. elevated
cholesterol, including total cholesterol, LDL cholesterol, and
triglyceride levels), hypertension, increased or decreased levels
of inflammatory mediators (for example, C-reactive protein,
homocysteine, and fibrinogen), aging (especially individuals over
50), racial background (especially prevalent among African-American
individuals), gender (more frequently seen in males), obesity, or
individuals with personal histories of vascular disease, heart
attack, or stroke. The present invention encompasses methods of
administering compositions, scaffolds, and devices to all
individuals listed supra, for the purposes of repairing or
replenishing blood supply to blood- and oxygen-deprived
tissues.
[0082] PAD/PAOD is diagnosed using a number of tests. The initial
test is an ankle brachial pressure index (ABPI/ABI) which measures
the fall in blood pressure in the arteries supplying the legs. A
reduced ABPI, quantitatively, a score of less than 0.9, suggests a
diagnosis of PAD/PAOD. Moderate PAD/PAOD is diagnosed with a
reduced ABPI score of less than 0.8. Severe PAD/PAOD is diagnosed
with a reduced ABPI score of less than 0.5. However, conditions
other than PAD/PAOD can result in reduced ABPI scores of less than
0.9. Thus, additional tests are performed to confirm a diagnosis of
PAD/PAOD. A secondary examination usually comprises a lower limb
Doppler ultrasound examination of the femoral artery.
Alternatively, or in addition, imaging examinations can be
performed by angiography using art-recognized standard methods.
Furthermore, a multi-slice computerized tomography (CT) scan is
used to directly image the arterial system.
[0083] PAD/PAOD severity is divided in the Fontaine stages
(Fontaine R, Kim M, Kieny R (1954). Helvetica Chirurgica Acta,
Basel 21 (5/6):499-533): mild pain while walking
("claudication")(stage I); severe pain on walking relatively
shorter distances (intermittent claudication)(stage II); pain while
resting (stage III); loss of sensation to the lower part of the
extremity (stage IV); tissue loss (gangrene)(stage V).
Angiogenic Bioactive Compositions
[0084] Compositions and methods of the present invention comprise
growth factors and signaling molecules that induce, regulate, or
augment angiogenesis. Bioactive compositions of the present
invention comprise one or more growth factors or signaling
molecules incorporated into or coated onto the scaffold
composition. Exemplary growth factors and signaling molecules
encompassed by the present invention include, but are not limited
to, vascular endothelial growth factor (VEGF (A-F)), fibroblast
growth factors (acidic and basic FGF 1-10), granulocyte-macrophage
colony-stimulating factor (GM-CSF), insulin, insulin growth factor
or insulin-like growth factor (IGF), insulin growth factor binding
protein (IGFBP), placenta growth factor (PIGF), angiopoietin (Ang1
and Ang2), platelet-derived growth factor (PDGF), hepatocyte growth
factor (HGF), transforming growth factor (TGF-.alpha., TGF-.beta.,
isoforms 1-3), platelet-endothelial cell adhesion molecule-1
(PECAM-1), vascular endothelial cadherin (VE-cadherin), nitric
oxide (NO), chemokine (C--X--C motif) ligand 10 (CXCL10) or IP-10,
interleukin-8 (IL-8), hypoxia inducible factor (HIF), monocyte
chemotactic protein (MCP), vascular cell adhesion molecule (VCAM),
ephrin ligands (including Ephrin-B2 and -B4); Transcription factors
such as HIF-1.alpha., HIF-1.beta. and HIF-2.alpha., Ets-1, Hex,
Vezf1, Hox, GATA, LKLF, COUP-TFII, Hox, MEF2, Braf, Prx-1, Prx-2,
CRP2/SmLIM and GATA family members, basic helix-loop-helix factors
and their inhibitors of differentiation; and regulatory molecules
include enzymes (matrix metalloproteinase (MMP), tissue plasminogen
activator (PLAT or tPA), cyclooxygenase (COX), angiogenin),
molecules regulating Notch signaling which consists of monoclonal
antibodies to Notch ligands and receptors, RNA interference,
antisense Notch, receptor and mastermind-like 1 (MAML1) decoys,
beta and gamma-secretase inhibitors (GSI), or any other molecules
that can activate or inhibit Notch signaling.
Vascular Endothelial Growth Factor (VEGF, Also Known as VEGF-A)
[0085] The term "VEGF" broadly encompasses two families of proteins
that result from the alternate splicing of a single gene, VEGF,
composed of 8 exons. The alternate splice sites reside in the exons
6, 7, and 8. However, the alternate splice site in the terminal
exon 8 is functionally important. One family of proteins arise from
the proximal splice site and are denoted (VEGF.sub.XXX). Proteins
produced by alternate splicing at this proximal location are
PRO-angiogenic and are expressed conditionally (for instance, when
tissues are hypoxic and secreted signals induce angiogenesis). The
other family of proteins arise from the distal splice site and are
denoted (VEGF.sub.XXXb). Proteins produced by alternate splicing at
this distal location are ANTI-angiogenic and are expressed in
healthy tissues under normal conditions.
[0086] VEGF exons 6 and 7 contain splice sites (result in the
inclusion or exclusion of exons 6 and 7) that affect heparin
binding affinity and amino acid number. Humans comprise
VEGF.sub.121, VEGF.sub.121b, VEGF.sub.145, VEGF.sub.165,
VEGF.sub.165b, VEGF.sub.189, and VEGF.sub.206. Heparin binding
affinity, interactions with heparin surface proteoglycans (HSPGs)
and neuropilin co-receptors on the cell surface mediated by amino
acid sequences in exons 6 and 7 enhance the ability of VEGF
variants to activate VEGF signaling receptors (VEGFRs).
[0087] Endogenous VEGF splice variants are released from cells as
glycosylated disulfide-bonded dimers. Structurally VEGF belongs to
the PDGF family of cysteine-knot growth factors comprising Placenta
growth factor (PIGF), VEGF-B, VEGF-C and VEGF-D (the VEGF
sub-family of growth factors). VEGF is sometimes referred to as
VEGF-A to differentiate it from these related growth factors. The
term "VEGF" used herein to describe the present invention is meant
to refer to VEGF-A.
[0088] Members of the VEGF family stimulate cellular responses by
binding to cell-surface tyrosine kinase receptors (the VEGFRs).
VEGF-A binds to VEGFR-1 (also known as Flt-1) and VEGFR-2 (also
known as KDR/Flk-1). VEGFR-2 is the predominant receptor for VEGF-A
mediating almost all of the known cellular responses to this growth
factor. The function of VEGFR-1 is unclear, although it is thought
to modulate VEGFR-2 signaling. VEGFR-1 may also sequester VEGF from
VEGFR-2 binding (which may be important during development).
[0089] Compositions, methods, and devices of the present invention
comprise all VEGF polypeptides generated from alternative splicing
including pro- and anti-angiogenic forms. Devices of the present
invention administered to a subject contain only pro-angiogenic
VEGF polypeptide splice forms. Alternatively, or in addition,
devices of the present invention administered to a subject contain
a mixture of pro- and anti-angiogenic VEGF polypeptide splice
forms. Pro- and anti-angiogenic VEGF polypeptide splice forms are
released by the scaffold composition of the device simultaneously
or sequentially. For example, the opposing splice forms are
released together in order to achieve a precise level of
stimulation. Alternatively, the opposing splice forms are released
sequentially to stimulate angiogenesis and subsequently attenuate
the signal when the desired result has been achieved. In another
embodiment, devices comprising pro-angiogenic VEGF polypeptide
splice forms are placed at the target tissue site while devices
comprising anti-angiogenic VEGF polypeptide splice forms are placed
in surrounding tissues in order to prevent pro-angiogenic signals
from disseminating into and stimulating non-target tissue.
[0090] Exemplary VEGF polypeptide splice forms comprised by the
compositions, methods, and devices of the present invention
include, but are not limited to, the polypeptides described by the
following sequences and SEQ ID NOs. VEGF polypeptide splice forms
are released from compositions, scaffolds, or devices of the
present invention as naked, or glycosylated polypeptides.
Alternatively, or in addition, VEGF polypeptide splice forms are
monomers or disulfide-bonded dimers. In a preferred embodiment,
VEGF polypeptide splice forms are released into target tissues from
compositions, scaffolds, and/or devices of the present invention as
glycosylated disulfide-bonded dimers.
[0091] Human VEGF.sub.148 is encoded by the following amino acid
sequence (NCBI Accession No. NP.sub.--001020540 and SEQ ID NO:
12):
TABLE-US-00001 1 mtdrqtdtap spsyhllpgr rrtvdaaasr gqgpepapgg
gvegvgargv alklfvqllg 61 csrfggavvr ageaepsgaa rsassgreep
qpeegeeeee keeergpqwr lgarkpgswt 121 geaavcadsa paarapqala
rasgrggrva rrgaeesgpp hspsrrgsas ragpgraset 181 mnfllswvhw
slalllylhh akwsqaapma egggqnhhev vkfmdvyqrs ychpietivd 241
ifqeypdeie yifkpscvpl mrcggccnde glecvptees nitmqimrik phqgqhigem
301 sflqhnkcec rpkkdrarqe npcgpcserr khlfvgdpqt ckcsckntds rckm
[0092] Human VEGF.sub.165 is encoded by the following amino acid
sequence (NCBI Accession No. NP.sub.--001020539 and SEQ ID NO:
13):
TABLE-US-00002 1 mtdrqtdtap spsyhllpgr rrtvdaaasr gqgpepapgg
gvegvgargv alklfvqllg 61 csrfggavvr ageaepsgaa rsassgreep
qpeegeeeee keeergpqwr lgarkpgswt 121 geaavcadsa paarapqala
rasgrggrva rrgaeesgpp hspsrrgsas ragpgraset 181 mnfllswvhw
slalllylhh akwsqaapma egggqnhhev vkfmdvyqrs ychpietlvd 241
ifqeypdeie yifkpscvpl mrcggccnde glecvptees nitmqimrik phqgqhigem
301 sflqhnkcec rpkkdrarqe npcgpcserr khlfvgdpqt ckcsckntds
rckarqleln 361 ertcrcdkpr r
[0093] Human VEGF.sub.165b is encoded by the following amino acid
sequence (NCBI Accession No. NP.sub.--001028928 and SEQ ID NO:
14):
TABLE-US-00003 1 mtdrqtdtap spsyhllpgr rrtvdaaasr gqgpepapgg
gvegvgargv alklfvqllg 61 csrfggavvr ageaepsgaa rsassgreep
qpeegeeeee keeergpqwr lgarkpgswt 121 geaavcadsa paarapqala
rasgrggrva rrgaeesgpp hspsrrgsas ragpgraset 181 mnfllswvhw
slalllylhh akwsqaapma egggqnhhev vkfmdvyqrs ychpietlvd 241
ifqeypdeie yifkpscvpl mrcggccnde glecvptees nitmqimrik phqgqhigem
301 sflqhnkcec rpkkdrarqe npcgpcserr khlfvgdpqt ckcsckntds
rckarqleln 361 ertcrsltrk d
[0094] Human VEGF.sub.183 is encoded by the following amino acid
sequence (NCBI Accession No. NP.sub.--001020538 and SEQ ID NO:
15):
TABLE-US-00004 1 mtdrqtdtap spsyhllpgr rrtvdaaasr gqgpepapgg
gvegvgargv alklfvqllg 61 csrfggavvr ageaepsgaa rsassgreep
qpeegeeeee keeergpqwr lgarkpgswt 121 geaavcadsa paarapqala
rasgrggrva rrgaeesgpp hspsrrgsas ragpgraset 181 mnfllswvhw
slalllylhh akwsqaapma egggqnhhev vkfmdvyqrs ychpietlvd 241
ifqeypdeie yifkpscvpl mrcggccnde glecvptees nitmqimrik phqgqhigem
301 sflqhnkcec rpkkdrarqe kksvrgkgkg qkrkrkksrp cgpcserrkh
lfvqdpqtck 361 csckntdsrc karqlelner tcrcdkprr
[0095] Human VEGF.sub.189 is encoded by the following amino acid
sequence (NCBI Accession No. NP.sub.--003367 and SEQ ID NO:
16):
TABLE-US-00005 1 mtdrqtdtap spsyhllpgr rrtvdaaasr gqgpepapgg
gvegvgargv alklfvqllg 61 csrfggavvr ageaepsgaa rsassgreep
qpeegeeeee keeergpqwr lgarkpgswt 121 geaavcadsa paarapqala
rasgrggrva rrgaeesgpp hspsrrgsas ragpgraset 181 mnfllswvhw
slalllylhh akwsqaapma egggqnhhev vkfmdvyqrs ychpietlvd 241
ifqeypdeie yifkpscvpl mrcggccnde glecvptees nitmqimrik phqgqhigem
301 sflqhnkcec rpkkdrarqe kksvrgkgkg qkrkrkksry kswsvpcgpc
serrkhlfvq 361 dpqtckcsck ntdsrckarq lelnertcrc dkprr
[0096] Human VEGF.sub.206 is encoded by the following amino acid
sequence (NCBI Accession No. NP.sub.--001020537 and SEQ ID NO:
17):
TABLE-US-00006 1 mtdrqtdtap spsyhllpgr rrtvdaaasr gqgpepapgg
gvegvgargv alklfvqllg 61 csrfggavvr ageaepsgaa rsassgreep
qpeegeeeee keeergpqwr lgarkpgswt 121 geaavcadsa paarapqala
rasgrggrva rrgaeesgpp hspsrrgsas ragpgraset 181 mnfllswvhw
slalllylhh akwsqaapma egggqnhhev vkfmdvyqrs ychpietlvd 241
ifqeypdeie yifkpscvpl mrcggccnde glecvptees nitmqimrik phqgqhigem
301 sflqhnkcec rpkkdrarqe kksvrgkgkg qkrkrkksry kswsvyvgar
cclmpwslpg 361 phpcgpcser rkhlfvqdpq tckcsckntd srckarqlel
nertcrcdkp rr
Gamma-Secretase Inhibitors (GSI)
[0097] Notch is a cell-surface receptor that regulates cell fate
decisions throughout development and under selected conditions in
adult tissues. Notch signaling results in widely variable outcomes
depending on the cells and signaling molecules involved. However,
it is generally known that binding of Notch ligands of the Delta
and Jagged families results in the proteolytic cleavage of Notch.
The Notch protein is first cleaved in the extracellular domain and
then subsequently cleaved in the transmembrane domain. The second
cleavage event is mediated by .gamma.-secretase. Notch cleavage
allows the intracellular domain of the receptor (the Notch
IntraCellular Domain, NICD) to translocate to the nucleus where it
regulates transcription. Thus, .gamma.-secretase is a Notch
activator.
[0098] Notch signaling is involved in angiogenesis and vascular
remodeling. Moreover, Notch signaling regulates endothelial cell
proliferation and migration events necessary to form new blood
vessels during angiogenesis in normal tissues as well as malignant
tumors. Methods of the present invention are drawn towards inducing
angiogenesis in normal tissues, not malignant tissues. Furthermore,
it is of great importance to avoid inducing a malignant state
within a stable or benign tumor by introducing pro-angiogenic
factors in the absence of factors to limit Notch activation. In one
preferred embodiment of the present invention, pro-angiogenic
factors are released from compositions, scaffolds, or devices,
either simultaneously or sequentially, with notch-inhibitors, e.g.
inhibitors of gamma-secretase (.gamma.-secretase), to prevent
stimulation of angiogenesis within neoplastic tissue.
[0099] Compositions, scaffolds, and devices of the present
invention comprise all inhibitors of Notch activation to be
released simultaneously or sequentially with pro-angiogenic
factors. Inhibitors of Notch activity encompassed by the present
invention block binding of one or more ligands to the Notch
receptor. Alternatively, or in addition, inhibitors of Notch
activity present intracellular signal transduction from the Notch
receptor or cleavage of the Notch receptor polypeptide. Notch
inhibitors of the present invention comprise endogenous or
exogenous small molecules, compounds, single- or double-stranded
RNA polynucleotides, single- or double-stranded DNA
polynucleotides, polypeptides, antibodies, intrabodies, natural or
synthetic ligands, genetically-engineered ligands, and
genetically-manipulated .gamma.-secretase proteins or fragments
thereof. Exemplary inhibitors of Notch activation include, but are
not limited to, monoclonal antibodies to Notch ligands and
receptors, RNA interference, antisense Notch, receptor and
mastermind-like 1 (MAML1) decoys, beta and gamma-secretase
inhibitors (GSI).
[0100] Gamma-secretase is an integral membrane protein that is one
part of a multi-subunit protease complex that cleaves single-pass
transmembrane proteins at residues within the transmembrane domain.
The gamma secretase complex comprises four individual proteins:
presenilin, nicastrin, APH-1 (anterior pharynx-defective 1), and
PEN-2 (presenilin enhancer 2). A fifth protein, known as CD147, is
a non-essential regulator of the complex whose absence increases
activity.
[0101] The proteins in the gamma secretase complex are heavily
modified by proteolysis during assembly and maturation of the
complex. Presenilin is an aspartyl protease comprising the
catalytic subunit and is activated by autocatalytic cleavage of to
N- and C-terminal fragments. Nicastrin maintains the stability of
the assembled complex, regulates intracellular protein trafficking,
and recognizes substrates via binding to the N-terminal ectodomain
of the target protein. PEN-2 associates with the complex via
binding of a transmembrane domain of presenilin and stabilizes the
complex after presenilin proteolysis. APH-1, which is required for
proteolytic activity, binds to the complex via a conserved alpha
helix interaction motif and aids in initiating assembly of
premature components.
[0102] The present invention comprises one or more inhibitors of
.gamma.-secretase which target one or more proteins of this complex
and inhibit one or more functions of these proteins. Alternatively,
or in addition, .gamma.-secretase inhibitors of the present
invention prevent assembly of the .gamma.-secretase protease
complex. Furthermore, contemplated .gamma.-secretase inhibitors of
the present invention inhibit intracellular signal transduction
from an assembled .gamma.-secretase protease complex. Contemplated
.gamma.-secretase inhibitors of the present invention bind one or
more proteins of the g-secretase complex and partially or entirely
block an activity or function. Exemplary .gamma.-secretase
inhibitors of the present invention decrease, prevent, or delay
activation, as well as inactivate one or more protease components.
Exemplary .gamma.-secretase inhibitors of the present invention
desensitize or down regulate the activity or expression of one or
more proteins of the .gamma.-secretase complex. Exemplary
.gamma.-secretase inhibitors of the present invention consist of,
consist essentially of, or comprise endogenous or exogenous small
molecules, compounds, single- or double-stranded RNA
polynucleotides, single- or double-stranded DNA polynucleotides,
polypeptides, antibodies, intrabodies, natural or synthetic
ligands, genetically-engineered ligands, and
genetically-manipulated .gamma.-secretase proteins or fragments
thereof.
[0103] Exemplary .gamma.-secretase inhibitors of the present
invention include, but are not limited to, DAPT and
N-[N-(3,5-Difluorophenacetyl-L-alanyl)]-S-phenylglycine t-butyl
ester.
Cell-Mediated Enzymatic Scaffold Degradation
[0104] Cells secrete enzymes that degrade the material of the
scaffold, thereby controlling the rate at which cells exit the
scaffold. Cells within close proximity to the implanted scaffold
composition secrete enzymes, such as collagenases and plasmin,
which degrade the polymer composition. This property is used in
certain embodiments to control the release of bioactive
compositions into the local cellular environment. The rate of
release of bioactive composition may thus be regulated by
controlling the density and susceptibility to these enzymes of
oligopeptides used as either cross-links in the material or as
components of the main chains. Certain materials are degraded in a
preprogrammed manner independent of cell action (e.g. hydrolytic
degradation of poly(lactide-co glycolide) as a degradable scaffold.
The scaffolds may be prepared such that the degradation time may be
controlled by using a mixture of degradable components in
proportions to achieve a desired degradation rate. Scaffold
compositions are sensitive to degradation by materials secreted by
the cells located immediately adjacent to the scaffold. One example
of this is the use of metalloproteinase (MMP)-sensitive substrate
in the scaffold matrix; bioactive composition is released when the
adjacent cells have secreted sufficient MMP to begin degradation of
the matrix.
Scaffold Compositions and Architecture
[0105] Components of the scaffolds are organized in a variety of
geometric shapes (e.g., beads, pellets), niches, planar layers
(e.g., thin sheets). For example, multicomponent scaffolds are
constructed in concentric layers each of which is characterized by
different physical qualities (% polymer, % crosslinking of polymer,
chemical composition of scaffold, pore size, porosity, and pore
architecture, stiffness, toughness, ductility, viscoelasticity, and
or composition of bioactive substances such as growth factors,
homing/migration factors, differentiation factors. Each niche has a
specific effect on a cell population, e.g., promoting or inhibiting
a specific cellular function, proliferation, differentiation,
elaboration of secreted factors or enzymes, or migration.
[0106] Cells incubated in the scaffold are educated and induced to
migrate out of the scaffold to directly affect a target tissue,
e.g., and injured tissue site. For example, stromal vascular cells
and smooth muscle cells are useful in sheet-like structures are
used for repair of vessel-like structures such as blood vessels or
layers of the body cavity. For example, such structures are used to
repair abdominal wall injuries or defects such as gastroschisis.
Similarly, sheet-like scaffolds seeded with dermal stem cells
and/or keratinocytes are used in bandages or wound dressings for
regeneration of dermal tissue. The device is placed or transplanted
on or next to a target tissue, in a protected location in the body,
next to blood vessels, or outside the body as in the case of an
external wound dressing. Devices are introduced into or onto a
bodily tissue using a variety of known methods and tools, e.g.,
spoon, tweezers or graspers, hypodermic needle, endoscopic
manipulator, endo- or trans-vascular-catheter, stereotaxic needle,
snake device, organ-surface-crawling robot (United States Patent
Application 20050154376; Ota et al., 2006, Innovations 1:227-231),
minimally invasive surgical devices, surgical implantation tools,
and transdermal patches. Devices can also be assembled in place,
for example by sequentially injecting or inserting matrix
materials. Scaffold devices are optionally recharged with cells or
with bioactive compounds, e.g., by sequential injection or spraying
of substances such as growth factors or differentiation
factors.
[0107] A scaffold or scaffold device is the physical structure upon
which or into which cells associate or attach, and a scaffold
composition is the material from which the structure is made. For
example, scaffold compositions include biodegradable or permanent
materials such as those listed below. The mechanical
characteristics of the scaffold vary according to the application
or tissue type for which regeneration is sought. It is
biodegradable (e.g., collagen, alginates, polysaccharides,
polyethylene glycol (PEG), poly(glycolide) (PGA), poly(L-lactide)
(PLA), or poly(lactide-co-glycolide) (PLGA) or permanent (e.g.,
silk). In the case of biodegradable structures, the composition is
degraded by physical or chemical action, e.g., level of hydration,
heat or ion exchange or by cellular action, e.g., elaboration of
enzyme, peptides, or other compounds by nearby or resident cells.
The consistency varies from a soft/pliable (e.g., a gel) to glassy,
rubbery, brittle, tough, elastic, stiff. The structures contain
pores, which are nanoporous, microporous, or macroporous, and the
pattern of the pores is optionally homogeneous, heterogenous,
aligned, repeating, or random.
[0108] Alginates are versatile polysaccharide based polymers that
may be formulated for specific applications by controlling the
molecular weight, rate of degradation and method of scaffold
formation. Coupling reactions can be used to covalently attach
bioactive epitopes, such as the cell adhesion sequence RGD to the
polymer backbone. Alginate polymers are formed into a variety of
scaffold types. Injectable hydrogels can be formed from low MW
alginate solutions upon addition of a cross-linking agent, such as
calcium ions, while macroporous scaffolds are formed by
lyophilization of high MW alginate discs. Differences in scaffold
formulation control the kinetics of scaffold degradation. Release
rates of morphogens or other bioactive substances from alginate
scaffolds are controlled by scaffold formulation to present
morphogens in a spatially and temporally controlled manner. This
controlled release not only eliminates systemic side effects and
the need for multiple injections, but can be used to create a
microenvironment that activates host cells at the implant site.
##STR00001##
[0109] The scaffold comprises a biocompatible polymer matrix that
is optionally biodegradable in whole or in part. A hydrogel is one
example of a suitable polymer matrix material. Examples of
materials which can form hydrogels include polylactic acid,
polyglycolic acid, PLGA polymers, alginates and alginate
derivatives, gelatin, collagen, agarose, natural and synthetic
polysaccharides, polyamino acids such as polypeptides particularly
poly(lysine), polyesters such as polyhydroxybutyrate and
poly-epsilon.-caprolactone, polyanhydrides; polyphosphazines,
poly(vinyl alcohols), poly(alkylene oxides) particularly
poly(ethylene oxides), poly(allylamines)(PAM), poly(acrylates),
modified styrene polymers such as poly(4-aminomethylstyrene),
pluronic polyols, polyoxamers, poly(uronic acids),
poly(vinylpyrrolidone) and copolymers of the above, including graft
copolymers.
[0110] The scaffolds are fabricated from a variety of synthetic
polymers and naturally-occurring polymers such as, but not limited
to, collagen, fibrin, hyaluronic acid, agarose, and laminin-rich
gels. One preferred material for the hydrogel is alginate or
modified alginate material. Alginate molecules are comprised of
(1-4)-linked .beta.-D-mannuronic acid (M units) and .alpha.
L-guluronic acid (G units) monomers, which can vary in proportion
and sequential distribution along the polymer chain. Alginate
polysaccharides are polyelectrolyte systems which have a strong
affinity for divalent cations (e.g. Ca.sup.+2, Mg.sup.+2,
Ba.sup.+2) and form stable hydrogels when exposed to these
molecules. See Martinsen A., et al., Biotech. & Bioeng., 33
(1989) 79-89.) For example, calcium cross-linked alginate hydrogels
are useful for dental applications, wound dressings chondrocyte
transplantation and as a matrix for other cell types.
[0111] An exemplary device utilizes an alginate or other
polysaccharide of a relatively low molecular weight, preferably of
size which, after dissolution, is at the renal threshold for
clearance by humans, e.g., the alginate or polysaccharide is
reduced to a molecular weight of 1000 to 80,000 daltons.
Preferably, the molecular mass is 1000 to 60,000 daltons,
particularly preferably 1000 to 50,000 daltons. It is also useful
to use an alginate material of high guluronate content since the
guluronate units, as opposed to the mannuronate units, provide
sites for ionic crosslinking through divalent cations to gel the
polymer. U.S. Pat. No. 6,642,363, incorporated herein by reference
discloses methods for making and using polymers containing
polysaccharides such as alginates or modified alginates that are
particularly useful for cell transplantation and tissue engineering
applications.
[0112] Useful polysaccharides other than alginates include agarose
and microbial polysaccharides such as those listed in the table
below.
TABLE-US-00007 Polysaccharide Scaffold Compositions Polymers.sup.a
Structure Fungal Pullulan (N) 1,4-; 1,6-.alpha.-D-Glucan
Scleroglucan (N) 1,3; 1,6-.alpha.-D-Glucan Chitin (N)
1,4-.beta.-D-Acetyl Glucosamine Chitosan (C)
1,4-.beta..-D-N-Glucosamine Elsinan (N) 1,4-;1,3-.alpha.-D-Glucan
Bacterial Xanthan gum (A) 1,4-.beta..-D-Glucan with D-mannose;
D-glucuronic Acid as side groups Curdlan (N) 1,3-.beta..-D-Glucan
(with branching) Dextran (N) 1,6-.alpha.-D-Glucan with some
1,2;1,3-; 1,4-.alpha.-linkages Gellan (A) 1,4-.beta..-D-Glucan with
rhamose, D-glucuronic acid Levan (N) 2,6-.beta.-D-Fructan with some
.beta.-2,1-branching Emulsan (A) Lipoheteropolysaccharide Cellulose
(N) 1,4-.beta.-D-Glucan .sup.aN-neutral, A = anionic and C =
cationic.
[0113] The scaffolds of the invention are porous or non-porous. For
example, the scaffolds are nanoporous having a diameter of less
than about 10 nm; microporous wherein the diameter of the pores are
preferably in the range of about 100 nm-20 .mu.m; or macroporous
wherein the diameter of the pores are greater than about 20 .mu.m,
more preferably greater than about 100 .mu.m and even more
preferably greater than about 400 .mu.m. In one example, the
scaffold is macroporous with aligned pores of about 400-500 .mu.m
in diameter. Other methods of preparing porous hydrogel products
are known in the art. (U.S. Pat. No. 6,511,650 incorporated herein
by reference).
Bioactive Compositions
[0114] The device includes one or more bioactive compositions.
Bioactive compositions are purified naturally-occurring,
synthetically produced, or recombinant compounds, e.g.,
polypeptides, nucleic acids, small molecules, or other agents. The
compositions described herein are purified. Purified compounds are
at least 60% by weight (dry weight) the compound of interest.
Preferably, the preparation is at least 75%, more preferably at
least 90%, and most preferably at least 99%, by weight the compound
of interest. Purity is measured by any appropriate standard method,
for example, by column chromatography, polyacrylamide gel
electrophoresis, or HPLC analysis.
[0115] The bioactive composition comprises an element to improve a
function of the scaffold composition or to promote angiogenesis.
For example, at least one cell adhesion molecule is incorporated
into or onto the polymer matrix to attach the scaffold composition
to the local tissue site and prevent diffusion of the device. Such
molecules are incorporated into the polymer matrix prior to
polymerization of the matrix or after polymerization of the matrix.
Examples of cell adhesion molecules include, but are not limited
to, peptides, proteins and polysaccharides. More specifically, cell
adhesion molecules include, but are not limited to, fibronectin,
laminin, collagen, thrombospondin 1, vitronectin, elastin,
tenascin, aggrecan, agrin, bone sialoprotein, cartilage matrix
protein, fibrinogen, fibrin, fibulin, mucins, entactin,
osteopontin, plasminogen, restrictin, serglycin, SPARC/osteonectin,
versican, von Willebrand Factor, polysaccharide heparin sulfate,
connexins, collagen, RGD (Arg-Gly-Asp) and YIGSR
(Tyr-Ile-Gly-Ser-Arg) (SEQ ID NO: 9) peptides and cyclic peptides,
glycosaminoglycans (GAGs), hyaluronic acid (HA),
condroitin-6-sulfate, integrin ligands, selectins, cadherins and
members of the immunoglobulin superfamily. Other examples include
neural cell adhesion molecules (NCAMs), intercellular adhesion
molecules (ICAMs), vascular cell adhesion molecule (VCAM-1),
platelet-endothelial cell adhesion molecule (PECAM-1), L1, and
CHL1.
[0116] Examples of some of these molecules and their function are
shown in the following table.
TABLE-US-00008 ECM Proteins and peptides and role in cell function
Seq. ID Protein Sequence No: Role Fibronectin RGDS Adhesion LDV
Adhesion REDV Adhesion Vitronectin RGDV Adhesion Laminin A LRGDN 7
Adhesion IKVAV 8 Neurite extension Laminin B1 YIGSR 9 Adhesion of
many cells, via 67 kD laminin receptor PDSGR 10 Adhesion Laminin B2
RNIAEIIKDA 11 Neurite extension Collagen 1 RGDT Adhesion of most
cells DGEA Adhesion of platelets, other cells Thrombospondin RGD
Adhesion of most cells VTXG Adhesion of platelets Hubbell, JA
(1995): Biomaterials in tissue engineering. Bio/Technology 13:
565-576. One-letter abbreviations of amino acids are used, X stands
for any amino acid.
[0117] Additional examples of suitable cell adhesion molecules are
shown below.
TABLE-US-00009 Amino acid sequences specific for proteoglycan
binding from extra- cellular matrix proteins SEQ. ID. SEQUENCE NO.
PROTEIN XBBXBX* 2 Consensus sequence PRRARV 3 Fibronectin
YEKPGSPPREVVPRPRPGV 4 Fibronectin RPSLAKKQRFRHRNRKGYRSQRGHSRGR 5
Vitronectin RIQNLLKITNLRIKFVK 6 Laminin
[0118] Particularly preferred cell adhesion molecules are peptides
or cyclic peptides containing the amino acid sequence
arginine-glycine-aspartic acid (RGD) which is known as a cell
attachment ligand and found in various natural extracellular matrix
molecules. A polymer matrix with such a modification provides cell
adhesion properties to the scaffold, and sustains long-term
survival of mammalian cell systems, as well as supporting cell
growth.
[0119] Coupling of the cell adhesion molecules to the polymer
matrix is accomplished using synthetic methods which are in general
known to one of ordinary skill in the art and are described in the
examples. Approaches to coupling of peptides to polymers are
discussed in Hirano and Mooney, Advanced Materials, p. 17-25
(2004). Other useful bonding chemistries include those discussed in
Hermanson, Bioconjugate Techniques, p. 152-185 (1996), particularly
by use of carbodiimide couplers, DCC and DIC (Woodward's Reagent
K). Since many of the cell adhesion molecules are peptides, they
contain a terminal amine group for such bonding. The amide bond
formation is preferably catalyzed by
1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), which is a
water soluble enzyme commonly used in peptide synthesis. The
density of cell adhesion ligands, a critical regulator of cellular
phenotype following adhesion to a biomaterial. (Massia and Hubbell,
J. Cell Biol. 114:1089-1100, 1991; Mooney et al., J. Cell Phys.
151:497-505, 1992; and Hansen et al., Mol. Biol. Cell 5:967-975,
1994) can be readily varied over a 5-order of magnitude density
range.
Device Construction
[0120] The scaffold structure is constructed out of a number of
different rigid, semi-rigid, flexible, gel, self-assembling, liquid
crystalline, or fluid compositions such as peptide polymers,
polysaccharides, synthetic polymers, hydrogel materials, ceramics
(e.g., calcium phosphate or hydroxyapatite), proteins,
glycoproteins, proteoglycans, metals and metal alloys. The
compositions are assembled into cell scaffold structures using
methods known in the art, e.g., injection molding, lyophilization
of preformed structures, printing, self-assembly, phase inversion,
solvent casting, melt processing, gas foaming, fiber
forming/processing, particulate leaching or a combination thereof.
The assembled devices are then implanted or administered to the
body of an individual to be treated.
[0121] The device is assembled in vivo in several ways. The
scaffold is made from a gelling material, which is introduced into
the body in its ungelled form where it gels in situ. Exemplary
methods of delivering device components to a site at which assembly
occurs include injection through a needle or other extrusion tool,
spraying, painting, or methods of deposit at a tissue site, e.g.,
delivery using an application device inserted through a cannula. In
one example, the ungelled or unformed scaffold material is mixed
with bioactive substances and cells prior to introduction into the
body or while it is introduced. The resultant in vivo/in situ
assembled scaffold contains a mixture of these substances and
cells.
[0122] In situ assembly of the scaffold occurs as a result of
spontaneous association of polymers or from synergistically or
chemically catalyzed polymerization. Synergistic or chemical
catalysis is initiated by a number of endogenous factors or
conditions at or near the assembly site, e.g., body temperature,
ions or pH in the body, or by exogenous factors or conditions
supplied by the operator to the assembly site, e.g., photons, heat,
electrical, sound, or other radiation directed at the ungelled
material after it has been introduced. The energy is directed at
the scaffold material by a radiation beam or through a heat or
light conductor, such as a wire or fiber optic cable or an
ultrasonic transducer. Alternatively, a shear-thinning material,
such as an amphiphile, is used which re-cross links after the shear
force exerted upon it, for example by its passage through a needle,
has been relieved.
[0123] Suitable hydrogels for both in vivo and ex vivo assembly of
scaffold devices are well known in the art and described, e.g., in
Lee et al., 2001, Chem. Rev. 7:1869-1879. The peptide amphiphile
approach to self-assembly assembly is described, e.g., in
Hartgerink et al., 2002, Proc. Natl. Acad. Sci. U.S.A.
99:5133-5138. A method for reversible gellation following shear
thinning is exemplified in Lee et al., 2003, Adv. Mat.
15:1828-1832.
[0124] A multiple compartment device is assembled in vivo by
applying sequential layers of similarly or differentially doped gel
or other scaffold material to the target site. For example, the
device is formed by sequentially injecting the next, inner layer
into the center of the previously injected material using a needle,
forming concentric spheroids. Non-concentric compartments are
formed by injecting material into different locations in a
previously injected layer. A multi-headed injection device extrudes
compartments in parallel and simultaneously. The layers are made of
similar or different scaffolding compositions differentially doped
with bioactive substances. Alternatively, compartments
self-organize based on their hydro-philic/phobic characteristics or
on secondary interactions within each compartment.
Compartmentalized Device
[0125] A compartmentalized device is designed and fabricated using
different compositions or concentrations of compositions for each
compartment. For example, a first bioactive composition is
encapsulated within hydrogels, using standard encapsulation
techniques (e.g., alginate microbead formation). This compartment
is then coated with a second layer of gel (e.g., double layered
alginate microbeads). This second compartment is formed from the
same material that contains bioactive composition elements, the
same material in a distinct form (e.g., varying mechanical
properties or porosity), or a completely different material that
provides appropriate chemical/physical properties.
[0126] Alternatively, the compartments are fabricated individually,
and then adhered to each other (e.g., a "sandwich" with an inner
compartment surrounded on one or all sides with the second
compartment). This latter construction approach is accomplished
using the intrinsic adhesiveness of each layer for the other,
diffusion and interpenetration of polymer chains in each layer,
polymerization or cross-linking of the second layer to the first,
use of an adhesive (e.g., fibrin glue), or physical entrapment of
one compartment in the other. The compartments self-assemble and
interface appropriately, either in vitro or in vivo, depending on
the presence of appropriate precursors (e.g., temperature sensitive
oligopeptides, ionic strength sensitive oligopeptides, block
polymers, cross-linkers and polymer chains (or combinations
thereof), and precursors containing cell adhesion molecules that
allow cell-controlled assembly). An individual with ordinary skill
in the art of stem cell biology and biomaterials can readily derive
a number of potentially useful designs for combining or separating
components of a bioactive composition.
[0127] Alternatively, the compartmentalized device is formed using
a printing technology. Successive layers of a scaffold precursor
doped with bioactive substances and/or cells is placed on a
substrate then cross linked, for example by self-assembling
chemistries. When the cross linking is controlled by chemical-,
photo- or heat-catalyzed polymerization, the thickness and pattern
of each layer is controlled by a masque, allowing complex three
dimensional patterns to be built up when un-cross-linked precursor
material is washed away after each catalyzation. (WT Brinkman et
al., Photo-cross-linking of type 1 collagen gels in the presence of
smooth muscle cells: mechanical properties, cell viability, and
function. Biomacromolecules, 2003 July-August; 4(4): 890-895.; W.
Ryu et al., The construction of three-dimensional micro-fluidic
scaffolds of biodegradable polymers by solvent vapor based bonding
of micro-molded layers. Biomaterials, 2007 February; 28(6):
1174-1184; Wright, Paul K. (2001). 21st Century manufacturing. New
Jersey: Prentice-Hall Inc.) Complex, multi-compartment layers are
also built up using an inkjet device which "paints" different
doped-scaffold precursors on different areas of the substrate.
Julie Phillippi (Carnegie Mellon University) presentation at the
annual meeting of the American Society for Cell Biology on Dec. 10,
2006; Print me a heart and a set of arteries, Aldhouse P., New
Scientist 13 Apr. 2006 Issue 2547 p 19.; Replacement organs, hot
off the press, C. Choi, New Scientist, 25 Jan. 2003, v2379. These
layers are built-up into complex, three dimensional compartments.
The device is also built using any of the following methods: Jetted
Photopolymer, Selective Laser Sintering, Laminated Object
Manufacturing, Fused Deposition Modeling, Single Jet Inkjet, Three
Dimensional Printing, or Laminated Object Manufacturing.
Growth Factors and Incorporation of Compositions into/onto a
Scaffold Device
[0128] Bioactive substances that influence growth, development,
movement, and other cellular functions are introduced into or onto
the scaffold structures. Such substances include BMP, bone
morphogenetic protein; ECM, extracellular matrix proteins or
fragments thereof; EGF, epidermal growth factor; FGF-2, fibroblast
growth factor 2; NGF, nerve growth factor; PDGF, platelet-derived
growth factor; PIGF, placental growth factor; TGF, transforming
growth factor, and VEGF, vascular endothelial growth factor.
Cell-cell adhesion molecules (cadherins, integrins, ALCAM, NCAM,
proteases) are optionally added to the scaffold composition.
[0129] Exemplary growth factors and ligands are provided in the
tables below.
TABLE-US-00010 Growth factors used for angiogenesis Growth factor
Abbreviation Relevant activities Vascular endothelial VEGF
Migration, proliferation and growth factor survival of ECs Basic
fibroblast bFGF-2 Migration, proliferation and growth factor
survival of ECs and many other cell types Platelet-derived PDGF
Promotes the maturation of blood growth factor vessels by the
recruitment of smooth muscle cells Angiopoietin-1 Ang-1 Strengthens
EC-smooth muscle cell interaction Angiopoietin-2 Ang-2 Weakens
EC-smooth muscle cell interaction Placental PIGF Stimulates
angiogenesis growth factor Transforming TGF Stabilizes new blood
vessels growth factor by promoting matrix deposition
TABLE-US-00011 Growth factors used for wound healing Growth Factor
Abbreviation Relevant activities Platelet-derived PDGF Active in
all stages of healing process growth factor Epidermal EGF Mitogenic
for keratinocytes growth factor Transforming TGF-.beta. Promotes
keratinocyte migration, ECM growth factor-.beta. synthesis and
remodeling, and differentiation of epithelial cells Fibroblast FGF
General stimulant for wound healing growth factor
TABLE-US-00012 Growth Factors Used for Tissue- Engineering Moleular
weight Representative supplier Growth factor Abbreviation (kDa)
Relevant activities of rH growth factor Epidermal growth EGF 6.2
Proliferation of epithelial, mesenchymal, PeproTech Inc. factor and
fibroblast cells (Rocky Hill, NJ, USA) Platelet-derived PDGF-AA
28.5 Proliferation and chemoattractant agent for PeproTech Inc.
growth factor PDGF-AB 25.5 smooth muscle cells; extracellular
matrix PDGF-BB 24.3 synthesis and deposition Transforming
TFG-.alpha. 5.5 Migration and proliferation of PeproTech Inc.
growth factor-.alpha. keratinocytes; extracellular matrix synthesis
and deposition Transforming TGF-.beta. 25.0 Proliferation and
differentiation of bone PeproTech Inc. growth factor-.beta. forming
cells; chemoattractant for fibroblasts Bone BMP-2 26.0
Differentiation and migration of bone Cell Sciences Inc.
morphogenetic BMP-7 31.5 forming cells (Norwood, MA, USA) protein
Basic fibroblast bFGF/FGF-2 17.2 Proliferation of fibroblasts and
initiation of PeproTech Inc. growth factor angiogenesis Vascular
endothelial VEGF.sub.165 38.2 Migration, proliferation, and
survival of PeproTech Inc. growth factor endothelial cells rH,
recombinant human
TABLE-US-00013 Immobilized ligands used in tissue engineering
Immobilized ligand* ECM molecule source Application RGD Multiple
ECM molecules, Enhance bone and cartilage tissue formation in
including fibronectin, vitro and in vivo vitronectin, laminin,
collagen Regulate neurite outgrowth in vitro and in vivo and
thrombospondin Promote myoblast adhesion, proliferation and
differentiation Enhance endothelial cell adhesion and proliferation
IKVAV (SEQ ID NO: 8), YIGSR Laminin Regulate neurite outgrowth in
vitro and in vivo (SEQ ID NO: 9), RNIAEIIKDI (SEQ ID NO: 11)
Recombinant fibronectin fragment Fibronectin Promote formulation of
focal contacts in pre- (FNIII.sub.7-10) osteoblasts
Ac-GCRDGPQ-GIWGQDRCG Common MMP substrates, Encourage cell-mediated
proteolytic degradation, (SEQ ID NO: 18) (e.g. collagen,
fibronectin, remodeling and bone regeneration (with RGD and
laminin) BMP-2 presentation) in vivo *Sequences are given in
single-letter amino acid code. MMP, matrix metalloproteinase.
[0130] The release profiles of bioactive substances from scaffold
devices is controlled by both factor diffusion and polymer
degradation, the dose of the factor loaded in the system, and the
composition of the polymer. Similarly, the range of action (tissue
distribution) and duration of action, or spatiotemporal gradients
of the released factors are regulated by these variables. The
diffusion and degradation of the factors in the tissue of interest
is optionally regulated by chemically modifying the factors (e.g.,
PEGylating growth factors). In both cases, the time frame of
release determines the time over which effective cell delivery by
the device is desired.
[0131] Carrier systems for tissue regeneration are described in the
table below.
TABLE-US-00014 Polymeric carriers used to deliver various growth
factors and the type of tissues regenerated Growth factor Carrier
Tissue regenerated EGF Gelatin Dermis PET suture Tendon PVA sponge
Dermis PDGF Chitosan-PLLA scaffold Craniofacial bone CMC gel Dermis
Fibrin Ligament Porous HA Long Bone TGF-.beta. Alginate Cartilage
PLA Long Bone CaP-titanium mesh Craniofacial bone Polyoxamer; PEO
gel Dermis rhBMP-2 Collagen sponge Long bone Craniofacial bone
HA-TCP granules Spinal bone HA-collagen Long bone PLA-DX-PEG
Ectopic and hip bone rHBMP-7 HA Spinal bone Collagen-CMC Spinal
bone Porous HA Craniofacial bone bFGF Chitosan Dermis
Heparin-alginate Blood vessels EVAc microspheres Blood vessels
Fibrin matrices Blood vessels VEGF PLG scaffold Blood vessels PLG
scaffold Blood vessels PLG microspheres Blood vessels Fibrin mesh
Blood vessels Abbreviations: PET, poly (ethylene terepthalate);
PVA, polyvinyl alcohol; PLLA, poly(L-lactic acid); CMC,
carboxymethylcellulose; HA, hydroxyapatite; PLA, poly(D,L-lactic
acid); CaP, calcium phosphate; PEO, poly (ethylene oxide); TCP,
tricalcium phosphate; PEG, poly(ethylene glycol); -DX-,
-p-dioxanone-; EVAc, ethylene vinyl acetate; PLG, poly
(lactide-co-glycolide).
[0132] The bioactive substances are added to the scaffold
compositions using known methods including surface absorption,
physical immobilization, e.g., using a phase change to entrap the
substance in the scaffold material. For example, a growth factor is
mixed with the scaffold composition while it is in an aqueous or
liquid phase, and after a change in environmental conditions (e.g.,
pH, temperature, ion concentration), the liquid gels or solidifies
thereby entrapping the bioactive substance. Alternatively, covalent
coupling, e.g., using alkylating or acylating agents, is used to
provide a stable, long-term presentation of a bioactive substance
on the scaffold in a defined conformation. Exemplary reagents for
covalent coupling of such substances are provided in the table
below.
TABLE-US-00015 Methods to covalently couple peptides/proteins to
polymers Functional Group Coupling reagents and Reacting groups on
of Polymer cross-linker proteins/peptides --OH Cyanogen bromide
(CNBr) --NH.sub.2 Cyanuric chloride 4-(4,6-Dimethoxy-1,3,5-triazin-
2-yl)-4-methyl-morpholinium chloride (DMT-MM) --NH.sub.2
Diisocyanate compounds --NH.sub.2 Diisothoncyanate compounds --OH
Glutaraldehyde Succinic anhydride --NH.sub.2 Nitrous Acid
--NH.sub.2 Hydrazine + nitrous acid --SH --Ph--OH --NH.sub.2
Carbodiimide compounds --COOH (e.g., EDC, DCC)[a] DMT-MM --COOH
Thionyl chloride --NH.sub.2 N-hydroxysuccinimide
N-hydroxysulfosuccinimide + EDC --SH Disulfide compound --SH
[a]EDC: 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide
hydrochloride; DCC: dicyclohexylcarbodiimide
[0133] The following materials and methods were used to generate
the data described herein.
Preparation and Loading of Polymer/Gel Compositions
[0134] Ultrapure alginates were purchased from ProNova Biomedical
(Norway). MVG alginate, a high-G-containing alginate (M/G ratio of
40/60 as specified by the manufacturer), was used as the high
molecular weight (molecular mass=250,000 Da) component to prepare
gels. Low molecular weight alginate (molecular mass=50,000 Da) was
obtained by gamma-irradiating high molecular weight alginate with a
cobalt-60 source for 4 h at a gamma-dose of 3.0 Mrad (Radiation
Lab, Massachusetts Institute of Technology). The alginate used to
form gels was a combination of the two different molecular weight
polymers at a ratio of 3:1. Both alginate polymers were diluted to
1% w/v in double-distilled H.sub.20, and 1% of the sugar residues
in the polymer chains were oxidized with sodium periodate (Aldrich,
St Louis, Mo.) by maintaining solutions in the dark for 17 h at
room temperature. An equimolar amount of ethylene glycol (Fisher,
Pittsburgh, Pa.) was added to stop the reaction, and the solution
was subsequently dialyzed (MWCO 1000, Spectra/Por, Rancho
Dominguez, Calif.) over 3 days. The solution was sterile filtered,
frozen (-20 degree C. overnight), lyophilized and stored at -20
degree C. To prepare gels, modified alginates were reconstituted in
EBM-2 (Lonza, Walkersville, Md.) to obtain a 2% w/v solution (75%
low molecular weight, 25% high molecular weight MVG used in all
experiments) prior to gelation. The 2% w/v alginate solutions were
cross-linked with aqueous slurries of a calcium sulfate solution
(0.21 g CaSO.sub.4 mL/L distilled H20) at a ratio of 25:1 (40
microliter of CaS04 per 1 mL of 2% w/v alginate solution) using a 1
mL syringe. Reconstituted alginate was stored at 4 degree C. For
incorporation of VEGF, PDGF and DAPT, alginate solutions were mixed
with recombinant human VEGF 165, PDGF-BB (R&D systems, MN) or
DAPT (N-[N-(3,5-Difluorophenacetyl-L-alanyl)]-Sphenylglycine
t-Butyl Ester) (EMD Chemicals, NJ) by using two syringes coupled by
a syringe connector. Calcium slurry (Sigma, St Louis, Mo.) was then
mixed with the resulting alginate solution using two syringes
coupled by a syringe connector to facilitate the mixing process and
prevent entrapment of air bubbles during mixing. The mixture was
allowed to gel for 30 min, and then was maintained at 4 degrees C.
prior to animal injections.
Murine Hindlimb Model of Ischemia
[0135] The animals used were 6-week old severe combined
immunodeficiency (SCID) mice on a C57BL/6J background (Jackson
Laboratory, ME). Unilateral hindlimb ischemia was created as
follows. The animals were anesthetized by intraperitoneal
injections of ketamine (80 mg/kg) and xylazine (5 mg/kg). The
external iliac and femoral artery and vein were ligated, and 50
.mu.L alginate hydrogel incorporating 3 .mu.g VEGF and/or 86-8600
ng DAPT was injected near the distal end of the ligation site. As
controls, VEGF and DAPT in PBS were also injected intramuscularly
or intraperitoneally (bolus delivery). Incisions were closed by 5-0
Ethicon sutures (Johnson & Johnson, NJ). Blood flow in the
hindlimb was monitored by a laser Doppler perfusion imaging (LDP/)
system (Perimed AB, Sweden), and the results were normalized to the
control unligated limb of the same animal.
Histology and Immunohistochemistry
[0136] Hindlimb muscle tissues between the two suture knots
defining the ligation site were dissected and fixed by Z-fix
(Anatech, M/) overnight and changed into 70% EtOH for storage prior
to histology processing. Samples were embedded in paraffin and
sectioned (5 .mu.m thick) onto slides by Paragon (Paragon
Bioservices, MD). Sections were incubated with primary anti-mouse
CD31 antibody (1:250) (Pharmingen, CA) or .alpha.-smooth muscle
actin antibody, followed by incubation with an anti-rat mouse
biotinylated secondary (1:200) (Vector Laboratories, CA), and
amplified by a Tyramide Signal Amplification (TSA) Biotin System
(Perkin Elmer Life Sciences, MA). Staining was developed using
DAB+substrate chromogen (DAKO, CA) and counterstained with Mayer's
Hematoxylin. Capillary densities were quantified by counting the
CD31 positive capillary numbers, normalized to the tissue area, in
30 randomly chosen high-power (200.times., 400.times.) fields.
Images were captured with an Olympus-IX81 light microscope
connected to an Olympus DP70 digital image capture system.
Example 1
In Vitro Model to Test the Significance of a Controlled Local
Concentration of VEGF
[0137] Endothelial cells isolated from diabetics are shown to have
a reduced response to VEGF as compared to age-matched non-diabetics
(FIG. 1, *P<0.05), as reflected in their capability of forming
sprouts, the first step in angiogenesis. In addition, there is an
optimal VEGF concentration to induce most sprouts. This suggests
the need of a controlled concentration of growth factors in the
local area to reach the best therapeutic effect.
Example 2
In Vitro Model to Test the Significance of a Combination of DAPT
and VEGF
[0138] Endothelial cells isolated from diabetics produce more
sprouts in the presence of a combination of both gamma-secretase
inhibitors with VEGF. (FIG. 2, *P<0.05) than with either of them
alone, which implies the need of a combination of both growth
factor and DAPT.
Example 3
In Vitro Model Establishing the Significance of a Distinct
Presentation of VEGF and DAPT
[0139] This examples illustrates the concept that while the
combination of VEGF and DAPT is superior to either single factor
alone, the optimal concentration for each individual compound does
not coincide. As shown in FIG. 3 (*P<0.05), the concentration of
VEGF that gives the most sprouts if used alone (50 ng/ml) is less
superior to a lower concentration (10 ng/ml) when in combined use
with DAPT (2.5 .mu.M). Moreover, increasing both the concentration
of VEGF (50 ng/ml) and DAPT (10 .mu.M) actually reduces the number
of sprouts. This indicates that the presentation of VEGF and DAPT
may need to be separately controlled to achieve an optimal
effect.
Example 4
In Vivo Model to Test the Effect of Controlled Presentation of VEGF
and DAPT to Recover Blood Flow in an Ischemia Situation
[0140] Alginate is used as the delivery vehicle. Injectable
alginate hydrogels incorporating VEGF and GSI is injected into the
hindlimb ischemia site created by femoral artery and vein ligation
in a murine model. Release profiles of VEGF and DAPT from alginate
hydrogels are distinct, as shown in FIG. 4A. Blood flow before and
after ligation surgery is measured by laser doppler perfusion
imaging (LDPI) to indicate the extent of angiogenesis in the
ischemia area. As shown in FIGS. 4B and 4C, (*P<0.05), when DAPT
and VEGF are both incorporated in the alginate hydrogel the blood
flow recovery is superior to simple bolus injection of them
together, either drug alone from alginate gel or by bolus injection
(including direct intramuscular and intraperitoneal injection), and
blank control. This points to the significance of a controlled
presentation of multiple pro-angiogenic compounds as compared to
bolus injection.
Example 5
In Vivo Model to Test the Effect of Controlled Presentation of VEGF
and DAPT on Newly-Formed Blood Vessel Density in an Ischemia
Situation
[0141] A combination of VEGF and DAPT delivered by alginate gel
systems gave rise to the highest blood vessel density as compared
to blank control, either alone released from gels (FIG. 5A), or
administered via intramuscular or intraperitoneal injection (FIG.
5B). These data indicate the significance of controlled delivery of
both VEGF and DAPT by the alginate gel system, i.e. controlled and
coordinated release of growth factors and signaling molecules leads
to an improved clinical result compared to conventional delivery
methods.
Example 6
In Vivo Evaluation of the Effect of DAPT Delivered from Alginate
Gel System and from Intraperitoneal Injection on the Cells in Small
Intestines
[0142] As compared to normal controls, DAPT delivered from alginate
gel system does not alter cell differentiation at distant sites as
much as DAPT delivered via intraperitoneal injection (DAPT
delivered via injection disrupts normal intestinal structure, as
indicated by H&E staining, presence of extra glycosaminoglycans
by alcian blue staining, loss of proliferating cells by Ki67
staining, and downregulation of Notch target gene expression by
HES-1 staining). DAPT delivered from the gel system only has
effects in the local region while DAPT delivered via
intraperitoneal injection goes into the systemic circulation and
led to adverse effects at distant organs (e.g., small intestines as
an example). These data indicate that a controlled local but not
systemic presentation of delivered DAPT is a preferred delivery
method.
Example 7
Blood Vessel Density and Blood Flow Recovery
[0143] In vivo evaluation of the effect of controlled presentation
of VEGF and DAPT on newly formed blood vessel density and blood
flow recovery was carried out in an ischemic type I diabetic mouse
model. The results (FIGS. 7A-B) indicate that a combination of
controlled delivery of a prescribed previously determined ratio,
e.g., an optimal level, of VEGF and DAPT increased the blood vessel
density and recovered blood flow.
Example 8
Comparison of Controlled Presentation of Factors Compared to
Delivery of Factors Alone
[0144] The effect of controlled presentation of VEGF and PDGF on
newly formed blood vessel density and blood flow recovery was
evaluated in vivo using the ischemic type I diabetic mouse model.
The results (FIGS. 8A-B) indicate that a combination of optimal
level of VEGF and PDGF is superior to VEGF or PDGF alone in
recovering blood flow.
Example 9
Controlled Presentation of a Combination of Factors VEGF, PDGF and
DAPT on Angiogenesis
[0145] In vivo evaluation of the effect of controlled presentation
of VEGF, PDGF and DAPT on newly formed blood vessel density and
blood flow recovery was carried out in an ischemic type I diabetic
mouse model. These results (FIGS. 9A-B) indicate that a combination
of optimal level of VEGF and DAPT is superior to a combination of
VEGF and PDGF in recovering blood flow.
Example 10
Maturation of Newly Formed Blood Vessels
[0146] The effect of controlled presentation of VEGF, PDGF and DAPT
on the maturation of newly formed blood vessels was evaluated using
the same in vivo ischemic type I diabetic mouse model. The result
(FIGS. 10A-C) indicates that a combination of optimal level of VEGF
PDGF and DAPT is superior to a combination of VEGF and PDGF in
generating more matured blood vessels.
[0147] The data generated described herein indicate that
compositions and methods not only reliably induce and promote
angiogenesis in bodily tissues and organs but also promote and
support maturation of those vessels into functional vasculature to
improve blood flow to ischemic, damaged, injured, or otherwise
compromised tissues and organs.
Other Embodiments
[0148] The patent and scientific literature referred to herein
establishes the knowledge that is available to those with skill in
the art. All United States patents and published or unpublished
United States patent applications cited herein are incorporated by
reference. All published foreign patents and patent applications
cited herein are hereby incorporated by reference. All other
published references, documents, manuscripts and scientific
literature cited herein are hereby incorporated by reference.
[0149] While this invention has been particularly shown and
described with references to preferred embodiments thereof, it will
be understood by those skilled in the art that various changes in
form and details may be made therein without departing from the
scope of the invention encompassed by the appended claims.
Sequence CWU 1
1
1819PRTHomo sapiens 1Gly Gly Gly Gly Arg Gly Asp Ser Pro 1 5
26PRTHomo sapiensmisc_feature(1)..(1)Xaa can be any naturally
occurring amino acid 2Xaa Asx Asx Xaa Asx Xaa 1 5 36PRTHomo sapiens
3Pro Arg Arg Ala Arg Val 1 5 419PRTHomo sapiens 4Tyr Glu Lys Pro
Gly Ser Pro Pro Arg Glu Val Val Pro Arg Pro Arg 1 5 10 15 Pro Gly
Val 528PRTHomo sapiens 5Arg Pro Ser Leu Ala Lys Lys Gln Arg Phe Arg
His Arg Asn Arg Lys 1 5 10 15 Gly Tyr Arg Ser Gln Arg Gly His Ser
Arg Gly Arg 20 25 617PRTHomo sapiens 6Arg Ile Gln Asn Leu Leu Lys
Ile Thr Asn Leu Arg Ile Lys Phe Val 1 5 10 15 Lys 75PRTHomo sapiens
7Leu Arg Gly Asp Asn 1 5 85PRTHomo sapiens 8Ile Lys Val Ala Val 1 5
95PRTHomo sapiens 9Tyr Ile Gly Ser Arg 1 5 105PRTHomo sapiens 10Pro
Asp Ser Gly Arg 1 5 1110PRTHomo sapiens 11Arg Asn Ile Ala Glu Ile
Ile Lys Asp Ala 1 5 10 12354PRTHomo sapiens 12Met Thr Asp Arg Gln
Thr Asp Thr Ala Pro Ser Pro Ser Tyr His Leu 1 5 10 15 Leu Pro Gly
Arg Arg Arg Thr Val Asp Ala Ala Ala Ser Arg Gly Gln 20 25 30 Gly
Pro Glu Pro Ala Pro Gly Gly Gly Val Glu Gly Val Gly Ala Arg 35 40
45 Gly Val Ala Leu Lys Leu Phe Val Gln Leu Leu Gly Cys Ser Arg Phe
50 55 60 Gly Gly Ala Val Val Arg Ala Gly Glu Ala Glu Pro Ser Gly
Ala Ala 65 70 75 80 Arg Ser Ala Ser Ser Gly Arg Glu Glu Pro Gln Pro
Glu Glu Gly Glu 85 90 95 Glu Glu Glu Glu Lys Glu Glu Glu Arg Gly
Pro Gln Trp Arg Leu Gly 100 105 110 Ala Arg Lys Pro Gly Ser Trp Thr
Gly Glu Ala Ala Val Cys Ala Asp 115 120 125 Ser Ala Pro Ala Ala Arg
Ala Pro Gln Ala Leu Ala Arg Ala Ser Gly 130 135 140 Arg Gly Gly Arg
Val Ala Arg Arg Gly Ala Glu Glu Ser Gly Pro Pro 145 150 155 160 His
Ser Pro Ser Arg Arg Gly Ser Ala Ser Arg Ala Gly Pro Gly Arg 165 170
175 Ala Ser Glu Thr Met Asn Phe Leu Leu Ser Trp Val His Trp Ser Leu
180 185 190 Ala Leu Leu Leu Tyr Leu His His Ala Lys Trp Ser Gln Ala
Ala Pro 195 200 205 Met Ala Glu Gly Gly Gly Gln Asn His His Glu Val
Val Lys Phe Met 210 215 220 Asp Val Tyr Gln Arg Ser Tyr Cys His Pro
Ile Glu Thr Leu Val Asp 225 230 235 240 Ile Phe Gln Glu Tyr Pro Asp
Glu Ile Glu Tyr Ile Phe Lys Pro Ser 245 250 255 Cys Val Pro Leu Met
Arg Cys Gly Gly Cys Cys Asn Asp Glu Gly Leu 260 265 270 Glu Cys Val
Pro Thr Glu Glu Ser Asn Ile Thr Met Gln Ile Met Arg 275 280 285 Ile
Lys Pro His Gln Gly Gln His Ile Gly Glu Met Ser Phe Leu Gln 290 295
300 His Asn Lys Cys Glu Cys Arg Pro Lys Lys Asp Arg Ala Arg Gln Glu
305 310 315 320 Asn Pro Cys Gly Pro Cys Ser Glu Arg Arg Lys His Leu
Phe Val Gln 325 330 335 Asp Pro Gln Thr Cys Lys Cys Ser Cys Lys Asn
Thr Asp Ser Arg Cys 340 345 350 Lys Met 13371PRTHomo sapiens 13Met
Thr Asp Arg Gln Thr Asp Thr Ala Pro Ser Pro Ser Tyr His Leu 1 5 10
15 Leu Pro Gly Arg Arg Arg Thr Val Asp Ala Ala Ala Ser Arg Gly Gln
20 25 30 Gly Pro Glu Pro Ala Pro Gly Gly Gly Val Glu Gly Val Gly
Ala Arg 35 40 45 Gly Val Ala Leu Lys Leu Phe Val Gln Leu Leu Gly
Cys Ser Arg Phe 50 55 60 Gly Gly Ala Val Val Arg Ala Gly Glu Ala
Glu Pro Ser Gly Ala Ala 65 70 75 80 Arg Ser Ala Ser Ser Gly Arg Glu
Glu Pro Gln Pro Glu Glu Gly Glu 85 90 95 Glu Glu Glu Glu Lys Glu
Glu Glu Arg Gly Pro Gln Trp Arg Leu Gly 100 105 110 Ala Arg Lys Pro
Gly Ser Trp Thr Gly Glu Ala Ala Val Cys Ala Asp 115 120 125 Ser Ala
Pro Ala Ala Arg Ala Pro Gln Ala Leu Ala Arg Ala Ser Gly 130 135 140
Arg Gly Gly Arg Val Ala Arg Arg Gly Ala Glu Glu Ser Gly Pro Pro 145
150 155 160 His Ser Pro Ser Arg Arg Gly Ser Ala Ser Arg Ala Gly Pro
Gly Arg 165 170 175 Ala Ser Glu Thr Met Asn Phe Leu Leu Ser Trp Val
His Trp Ser Leu 180 185 190 Ala Leu Leu Leu Tyr Leu His His Ala Lys
Trp Ser Gln Ala Ala Pro 195 200 205 Met Ala Glu Gly Gly Gly Gln Asn
His His Glu Val Val Lys Phe Met 210 215 220 Asp Val Tyr Gln Arg Ser
Tyr Cys His Pro Ile Glu Thr Leu Val Asp 225 230 235 240 Ile Phe Gln
Glu Tyr Pro Asp Glu Ile Glu Tyr Ile Phe Lys Pro Ser 245 250 255 Cys
Val Pro Leu Met Arg Cys Gly Gly Cys Cys Asn Asp Glu Gly Leu 260 265
270 Glu Cys Val Pro Thr Glu Glu Ser Asn Ile Thr Met Gln Ile Met Arg
275 280 285 Ile Lys Pro His Gln Gly Gln His Ile Gly Glu Met Ser Phe
Leu Gln 290 295 300 His Asn Lys Cys Glu Cys Arg Pro Lys Lys Asp Arg
Ala Arg Gln Glu 305 310 315 320 Asn Pro Cys Gly Pro Cys Ser Glu Arg
Arg Lys His Leu Phe Val Gln 325 330 335 Asp Pro Gln Thr Cys Lys Cys
Ser Cys Lys Asn Thr Asp Ser Arg Cys 340 345 350 Lys Ala Arg Gln Leu
Glu Leu Asn Glu Arg Thr Cys Arg Cys Asp Lys 355 360 365 Pro Arg Arg
370 14371PRTHomo sapiens 14Met Thr Asp Arg Gln Thr Asp Thr Ala Pro
Ser Pro Ser Tyr His Leu 1 5 10 15 Leu Pro Gly Arg Arg Arg Thr Val
Asp Ala Ala Ala Ser Arg Gly Gln 20 25 30 Gly Pro Glu Pro Ala Pro
Gly Gly Gly Val Glu Gly Val Gly Ala Arg 35 40 45 Gly Val Ala Leu
Lys Leu Phe Val Gln Leu Leu Gly Cys Ser Arg Phe 50 55 60 Gly Gly
Ala Val Val Arg Ala Gly Glu Ala Glu Pro Ser Gly Ala Ala 65 70 75 80
Arg Ser Ala Ser Ser Gly Arg Glu Glu Pro Gln Pro Glu Glu Gly Glu 85
90 95 Glu Glu Glu Glu Lys Glu Glu Glu Arg Gly Pro Gln Trp Arg Leu
Gly 100 105 110 Ala Arg Lys Pro Gly Ser Trp Thr Gly Glu Ala Ala Val
Cys Ala Asp 115 120 125 Ser Ala Pro Ala Ala Arg Ala Pro Gln Ala Leu
Ala Arg Ala Ser Gly 130 135 140 Arg Gly Gly Arg Val Ala Arg Arg Gly
Ala Glu Glu Ser Gly Pro Pro 145 150 155 160 His Ser Pro Ser Arg Arg
Gly Ser Ala Ser Arg Ala Gly Pro Gly Arg 165 170 175 Ala Ser Glu Thr
Met Asn Phe Leu Leu Ser Trp Val His Trp Ser Leu 180 185 190 Ala Leu
Leu Leu Tyr Leu His His Ala Lys Trp Ser Gln Ala Ala Pro 195 200 205
Met Ala Glu Gly Gly Gly Gln Asn His His Glu Val Val Lys Phe Met 210
215 220 Asp Val Tyr Gln Arg Ser Tyr Cys His Pro Ile Glu Thr Leu Val
Asp 225 230 235 240 Ile Phe Gln Glu Tyr Pro Asp Glu Ile Glu Tyr Ile
Phe Lys Pro Ser 245 250 255 Cys Val Pro Leu Met Arg Cys Gly Gly Cys
Cys Asn Asp Glu Gly Leu 260 265 270 Glu Cys Val Pro Thr Glu Glu Ser
Asn Ile Thr Met Gln Ile Met Arg 275 280 285 Ile Lys Pro His Gln Gly
Gln His Ile Gly Glu Met Ser Phe Leu Gln 290 295 300 His Asn Lys Cys
Glu Cys Arg Pro Lys Lys Asp Arg Ala Arg Gln Glu 305 310 315 320 Asn
Pro Cys Gly Pro Cys Ser Glu Arg Arg Lys His Leu Phe Val Gln 325 330
335 Asp Pro Gln Thr Cys Lys Cys Ser Cys Lys Asn Thr Asp Ser Arg Cys
340 345 350 Lys Ala Arg Gln Leu Glu Leu Asn Glu Arg Thr Cys Arg Ser
Leu Thr 355 360 365 Arg Lys Asp 370 15389PRTHomo sapiens 15Met Thr
Asp Arg Gln Thr Asp Thr Ala Pro Ser Pro Ser Tyr His Leu 1 5 10 15
Leu Pro Gly Arg Arg Arg Thr Val Asp Ala Ala Ala Ser Arg Gly Gln 20
25 30 Gly Pro Glu Pro Ala Pro Gly Gly Gly Val Glu Gly Val Gly Ala
Arg 35 40 45 Gly Val Ala Leu Lys Leu Phe Val Gln Leu Leu Gly Cys
Ser Arg Phe 50 55 60 Gly Gly Ala Val Val Arg Ala Gly Glu Ala Glu
Pro Ser Gly Ala Ala 65 70 75 80 Arg Ser Ala Ser Ser Gly Arg Glu Glu
Pro Gln Pro Glu Glu Gly Glu 85 90 95 Glu Glu Glu Glu Lys Glu Glu
Glu Arg Gly Pro Gln Trp Arg Leu Gly 100 105 110 Ala Arg Lys Pro Gly
Ser Trp Thr Gly Glu Ala Ala Val Cys Ala Asp 115 120 125 Ser Ala Pro
Ala Ala Arg Ala Pro Gln Ala Leu Ala Arg Ala Ser Gly 130 135 140 Arg
Gly Gly Arg Val Ala Arg Arg Gly Ala Glu Glu Ser Gly Pro Pro 145 150
155 160 His Ser Pro Ser Arg Arg Gly Ser Ala Ser Arg Ala Gly Pro Gly
Arg 165 170 175 Ala Ser Glu Thr Met Asn Phe Leu Leu Ser Trp Val His
Trp Ser Leu 180 185 190 Ala Leu Leu Leu Tyr Leu His His Ala Lys Trp
Ser Gln Ala Ala Pro 195 200 205 Met Ala Glu Gly Gly Gly Gln Asn His
His Glu Val Val Lys Phe Met 210 215 220 Asp Val Tyr Gln Arg Ser Tyr
Cys His Pro Ile Glu Thr Leu Val Asp 225 230 235 240 Ile Phe Gln Glu
Tyr Pro Asp Glu Ile Glu Tyr Ile Phe Lys Pro Ser 245 250 255 Cys Val
Pro Leu Met Arg Cys Gly Gly Cys Cys Asn Asp Glu Gly Leu 260 265 270
Glu Cys Val Pro Thr Glu Glu Ser Asn Ile Thr Met Gln Ile Met Arg 275
280 285 Ile Lys Pro His Gln Gly Gln His Ile Gly Glu Met Ser Phe Leu
Gln 290 295 300 His Asn Lys Cys Glu Cys Arg Pro Lys Lys Asp Arg Ala
Arg Gln Glu 305 310 315 320 Lys Lys Ser Val Arg Gly Lys Gly Lys Gly
Gln Lys Arg Lys Arg Lys 325 330 335 Lys Ser Arg Pro Cys Gly Pro Cys
Ser Glu Arg Arg Lys His Leu Phe 340 345 350 Val Gln Asp Pro Gln Thr
Cys Lys Cys Ser Cys Lys Asn Thr Asp Ser 355 360 365 Arg Cys Lys Ala
Arg Gln Leu Glu Leu Asn Glu Arg Thr Cys Arg Cys 370 375 380 Asp Lys
Pro Arg Arg 385 16395PRTHomo sapiens 16Met Thr Asp Arg Gln Thr Asp
Thr Ala Pro Ser Pro Ser Tyr His Leu 1 5 10 15 Leu Pro Gly Arg Arg
Arg Thr Val Asp Ala Ala Ala Ser Arg Gly Gln 20 25 30 Gly Pro Glu
Pro Ala Pro Gly Gly Gly Val Glu Gly Val Gly Ala Arg 35 40 45 Gly
Val Ala Leu Lys Leu Phe Val Gln Leu Leu Gly Cys Ser Arg Phe 50 55
60 Gly Gly Ala Val Val Arg Ala Gly Glu Ala Glu Pro Ser Gly Ala Ala
65 70 75 80 Arg Ser Ala Ser Ser Gly Arg Glu Glu Pro Gln Pro Glu Glu
Gly Glu 85 90 95 Glu Glu Glu Glu Lys Glu Glu Glu Arg Gly Pro Gln
Trp Arg Leu Gly 100 105 110 Ala Arg Lys Pro Gly Ser Trp Thr Gly Glu
Ala Ala Val Cys Ala Asp 115 120 125 Ser Ala Pro Ala Ala Arg Ala Pro
Gln Ala Leu Ala Arg Ala Ser Gly 130 135 140 Arg Gly Gly Arg Val Ala
Arg Arg Gly Ala Glu Glu Ser Gly Pro Pro 145 150 155 160 His Ser Pro
Ser Arg Arg Gly Ser Ala Ser Arg Ala Gly Pro Gly Arg 165 170 175 Ala
Ser Glu Thr Met Asn Phe Leu Leu Ser Trp Val His Trp Ser Leu 180 185
190 Ala Leu Leu Leu Tyr Leu His His Ala Lys Trp Ser Gln Ala Ala Pro
195 200 205 Met Ala Glu Gly Gly Gly Gln Asn His His Glu Val Val Lys
Phe Met 210 215 220 Asp Val Tyr Gln Arg Ser Tyr Cys His Pro Ile Glu
Thr Leu Val Asp 225 230 235 240 Ile Phe Gln Glu Tyr Pro Asp Glu Ile
Glu Tyr Ile Phe Lys Pro Ser 245 250 255 Cys Val Pro Leu Met Arg Cys
Gly Gly Cys Cys Asn Asp Glu Gly Leu 260 265 270 Glu Cys Val Pro Thr
Glu Glu Ser Asn Ile Thr Met Gln Ile Met Arg 275 280 285 Ile Lys Pro
His Gln Gly Gln His Ile Gly Glu Met Ser Phe Leu Gln 290 295 300 His
Asn Lys Cys Glu Cys Arg Pro Lys Lys Asp Arg Ala Arg Gln Glu 305 310
315 320 Lys Lys Ser Val Arg Gly Lys Gly Lys Gly Gln Lys Arg Lys Arg
Lys 325 330 335 Lys Ser Arg Tyr Lys Ser Trp Ser Val Pro Cys Gly Pro
Cys Ser Glu 340 345 350 Arg Arg Lys His Leu Phe Val Gln Asp Pro Gln
Thr Cys Lys Cys Ser 355 360 365 Cys Lys Asn Thr Asp Ser Arg Cys Lys
Ala Arg Gln Leu Glu Leu Asn 370 375 380 Glu Arg Thr Cys Arg Cys Asp
Lys Pro Arg Arg 385 390 395 17412PRTHomo sapiens 17Met Thr Asp Arg
Gln Thr Asp Thr Ala Pro Ser Pro Ser Tyr His Leu 1 5 10 15 Leu Pro
Gly Arg Arg Arg Thr Val Asp Ala Ala Ala Ser Arg Gly Gln 20 25 30
Gly Pro Glu Pro Ala Pro Gly Gly Gly Val Glu Gly Val Gly Ala Arg 35
40 45 Gly Val Ala Leu Lys Leu Phe Val Gln Leu Leu Gly Cys Ser Arg
Phe 50 55 60 Gly Gly Ala Val Val Arg Ala Gly Glu Ala Glu Pro Ser
Gly Ala Ala 65 70 75 80 Arg Ser Ala Ser Ser Gly Arg Glu Glu Pro Gln
Pro Glu Glu Gly Glu 85 90 95 Glu Glu Glu Glu Lys Glu Glu Glu Arg
Gly Pro Gln Trp Arg Leu Gly 100 105 110 Ala Arg Lys Pro Gly Ser Trp
Thr Gly Glu Ala Ala Val Cys Ala Asp 115 120 125 Ser Ala Pro Ala Ala
Arg Ala Pro Gln Ala Leu Ala Arg Ala Ser Gly 130 135 140 Arg Gly Gly
Arg Val Ala Arg Arg Gly Ala Glu Glu Ser Gly Pro Pro 145 150 155 160
His Ser Pro Ser Arg Arg Gly Ser Ala Ser Arg Ala Gly Pro Gly Arg 165
170 175 Ala Ser Glu Thr Met Asn Phe Leu Leu Ser Trp Val His Trp Ser
Leu 180 185 190 Ala Leu Leu Leu Tyr Leu His His Ala Lys Trp Ser Gln
Ala Ala Pro 195 200 205 Met Ala Glu Gly Gly Gly Gln Asn His His Glu
Val Val Lys Phe Met 210 215 220 Asp Val Tyr Gln Arg Ser Tyr Cys His
Pro Ile Glu Thr Leu Val Asp 225 230 235 240 Ile Phe Gln Glu Tyr Pro
Asp Glu Ile Glu Tyr Ile Phe Lys Pro Ser 245 250 255 Cys Val Pro Leu
Met Arg Cys Gly Gly Cys
Cys Asn Asp Glu Gly Leu 260 265 270 Glu Cys Val Pro Thr Glu Glu Ser
Asn Ile Thr Met Gln Ile Met Arg 275 280 285 Ile Lys Pro His Gln Gly
Gln His Ile Gly Glu Met Ser Phe Leu Gln 290 295 300 His Asn Lys Cys
Glu Cys Arg Pro Lys Lys Asp Arg Ala Arg Gln Glu 305 310 315 320 Lys
Lys Ser Val Arg Gly Lys Gly Lys Gly Gln Lys Arg Lys Arg Lys 325 330
335 Lys Ser Arg Tyr Lys Ser Trp Ser Val Tyr Val Gly Ala Arg Cys Cys
340 345 350 Leu Met Pro Trp Ser Leu Pro Gly Pro His Pro Cys Gly Pro
Cys Ser 355 360 365 Glu Arg Arg Lys His Leu Phe Val Gln Asp Pro Gln
Thr Cys Lys Cys 370 375 380 Ser Cys Lys Asn Thr Asp Ser Arg Cys Lys
Ala Arg Gln Leu Glu Leu 385 390 395 400 Asn Glu Arg Thr Cys Arg Cys
Asp Lys Pro Arg Arg 405 410 1816PRTHomo
sapiensMOD_RES(1)..(1)ACETYLATION 18Gly Cys Arg Asp Gly Pro Gln Gly
Ile Trp Gly Gln Asp Arg Cys Gly 1 5 10 15
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